Methods of rational nicotine hapten design and uses thereof

ABSTRACT

Provided herein are methods for rational design of nicotine haptens. More particularly, provided herein are methods for designing, selecting, and synthesizing nicotine haptens and nicotine hapten conjugates. Also provided herein are novel nicotine haptens and methods for using nicotine haptens to treat nicotine addiction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/189,930, filed Jun. 22, 2016, which claims the benefit of U.S.Provisional Patent Application No. 62/183,445, filed Jun. 23, 2015, eachof which is incorporated herein by reference as if set forth in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 DA035554awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to methods for rationally designingnicotine haptens. More particularly, the present invention relates tothe design process, to the selection and synthesis of nicotine haptensand nicotine hapten conjugates, and to their uses for nicotine vaccines.

2. Background

Cigarette smoking causes various types of diseases with high morbiditiesand mortality, but smoking cessation is difficult due to theaddictiveness of nicotine. Nicotine molecules enter the brain withinseconds after smoke inhalation, where it triggers the brain's responseto rewarding stimuli and consequently establishment of addiction.Despite extensive efforts and resources directed toward the developmentof cessation interventions, smoking remains a major public healthproblem. Nicotine antibodies have preventive and therapeutic potentialto curb this addictive behavior. Nicotine antibodies bind to nicotine inthe bloodstream and decrease the amount of nicotine that reaches thebrain. Passive transfer of nicotine-specific monoclonal antibodiesoffers control over nicotine antibody dose, but this therapeuticapproach is expensive and has a relative short duration of action.

Nicotine vaccines have emerged as a potentially promising treatment toreduce tobacco dependence by eliciting the production of anti-nicotineantibodies that can block nicotine transfer into the brain. Standardnicotine vaccines are based on conjugates comprising a nicotine haptenlinked to a protein. Nicotine haptens using linkers of various lengthsand rigidities have been created, but few have been shown tosignificantly improve anti-nicotine immune responses and, to date,clinical trials of nicotine vaccines have been unsuccessful. Therefore,there remains a need in the art for improved methods for designingnicotine haptens that the production of anti-nicotine antibodies andimproved therapeutic methods for treating nicotine addiction.

SUMMARY OF THE INVENTION

In a first aspect, provided herein is a method for obtaining a nicotinehapten capable of eliciting an immune response specific to nicotine. Themethod can comprise or consist essentially of (a) providingthree-dimensional structural information of an immunogenic carrier; (b)selecting functional groups or small molecule fragments predicted tobind to free nicotine at an immunogenic carrier binding site, wherein afunctional group or small molecule fragment is selected if indicated toexhibit higher binding energy for free nicotine than for the immunogeniccarrier; and (c) linking the selected functional group or small moleculefragment in a single compound, wherein the compound is a nicotine haptenthat, when conjugated to the immunogenic carrier, elicits production ofanti-nicotine antibodies having an affinity index greater than 0.02 andless than or equal to 1. Selecting can comprise using a computer havinga non-transitory computer-readable storage medium containing programmingto perform a fitting operation and to determine one or more bindingenergy parameters between nicotine and a binding site of the immunogeniccarrier. The method can further comprise analyzing results of thefitting operation to characterize the association between nicotine andthe binding pocket. The method can further comprise synthesizing orobtaining the compound; and evaluating the compound for its ability tocompete with free nicotine for binding. Evaluating can compriseperforming an indirect competitive enzyme-linked immunosorbent assay(ELISA). The hapten can comprise nicotine or a nicotine derivative. Theimmunogenic carrier can be a streptavidin.

In another aspect, provided herein is a nicotine hapten comprising astructure selected from the group consisting of:

In another aspect, provided herein is a nicotine hapten conjugatecomprising a nicotine hapten and an immunogenic carrier linked to thenicotine hapten, wherein the conjugate is an immunogen capable ofeliciting production of antibodies having an affinity index greater than0.02 and less than or equal to 1.

The nicotine hapten can comprise nicotine or a nicotine derivative. Thenicotine hapten can have a structure selected from the group consistingof:

The nicotine hapten can have the structure:

In another aspect, provided herein is a therapeutic composition capableof eliciting an immune response to nicotine, the composition comprisingat least one nicotine hapten conjugate as provided herein. Thecomposition can further comprise an adjuvant bound to the nicotinehapten conjugate.

In another aspect, provided herein is a method for eliciting an immuneresponse in a subject, the method comprising administering to thesubject a composition as provided herein.

These and other features, aspects, and advantages of the presentinvention will become better understood upon consideration of thefollowing detailed description, drawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of exemplary nicotine haptens.

FIG. 2 shows a scheme of hapten synthesis from 5-bromonicotinic acid.

FIG. 3 shows hapten synthesis schemes for compounds 10-21.

FIG. 4 shows hapten synthesis schemes for compounds 22-29.

FIG. 5 shows hapten synthesis schemes for compounds 30-33.

FIG. 6 shows hapten synthesis schemes for compounds 34-41.

FIG. 7 shows hapten synthesis schemes for compounds 42-54.

FIG. 8 is a table presenting binding affinity data (IC₅₀) for nicotine(Nic)-specific monoclonal antibodies (mAb) to nicotine haptens (Nic-HP)versus free nicotine.

FIGS. 9A-9C are graphs representing molecular interactions betweennicotine haptens and streptavidin to rank the accessibility ofconjugated haptens to a Nic-specific antibody. The binding energy(kcal/mol) values predicted using the AutoDock4 docking algorithm forthe interactions between nicotine haptens and streptavidin were averagedfor four lysine positions of streptavidin and plotted in the order ofnicotine haptens having higher binding energy considered better in thisstudy to lower binding energy. (A) The lowest binding energyconformation of nicotine haptens towards streptavidin irrespective ofthe presence of conformations with expected conjugation, whereas (B) thebinding energy of those which showed the expected conjugatableconformation of nicotine haptens on to streptavidin and (C) the bindingenergy of nicotine haptens that showed expected conjugatableconformation at all four lysine positions on streptavidin.

FIG. 10 is a table presenting relative binding activities of mAb toNic-HP versus nicotine-streptavidin conjugates (Nic-SA). Half inhibitoryconcentration (IC₅₀) of each hapten or free nicotine is determined bycompetition ELISA. The ratios between hapten IC₅₀ (IC_(50HP)) and freenicotine IC₅₀ (IC_(50Nic)) is defined as relative affinity index andcalculated to compare the binding activity of various haptens to thenicotine-specific monoclonal antibody 5F3. The ratios between IC_(50HP)and hapten-streptavidin IC₅₀ (IC_(50HP-SA)) is calculated to estimatethe effect of SA on the interaction between hapten and monoclonalantibody 5F3.

FIG. 11 is data for anti-nicotine antibody levels in mouse serum. Balb/cmice were immunized s.c. with various hapten-SA-CpG vaccines for threetimes and blood was collected from mouse cheek vein at 40 days post thethird immunization. Serum anti-nicotine antibody titer is analyzed byELISA. Nunc Maxisorp 96-well plates were coated with individualhapten-conjugated BSA, and the monoclonal antibody 5F3 is included asstandard on each plate. Antibody titer is defined as the dilution foldof each serum when the absorbance reaches to the half maximum absorbanceof 5F3 standard. Each dot in the graph represents one mouse, and themean titer of each group is shown as a bar. Group 138 and 140 showshigher anti-nicotine titer compared to 137 and 1′ groups.

FIG. 12 is a table demonstrating changes of anti-nicotine antibody leveland relative binding affinity to free nicotine in mouse serum at 15 daysafter second and 40 days post third immunization. Mice were immunizeds.c. with hapten-SA-CpG vaccines three times. Anti-nicotine antibodytiter and binding affinity of serum to free nicotine were measured byELISA and competition ELISA, respectively, after second and thirdimmunization. The quality index is defined as the ratio between titerand corresponding IC₅₀ (μM) as a parameter to compare the antibodyquality. Both anti-nicotine titers and binding affinity in all threegroups increases after third immunization compared to that after secondimmunization. Group of 140-SA-CpG is highlighted to emphasize thedrastic increase of antibody quality induced by 140 hapten-SA.

FIG. 13 demonstrates functional efficacy of nicotine vaccines ascharacterized by the distribution of s.c. injected nicotine in mouseserum and brain tissue (n=5). Ten days after the final challenge withhapten-SA, mice were injected s.c. with 0.1 mg/kg nicotine dissolved inPBS. Mice were decapitated 4 min later, and the brain and blood werecollected for nicotine concentration analysis by gas chromatography.Brain nicotine concentrations were corrected for brain blood content.Non-immunized mice were included as naïve control groups. * representsp<0.05, *** represents p<0.001, and **** represents p<0.0001 compared tonaïve mice, as measured by one-way ANOVA with Dunnett's correction.

FIG. 14 demonstrates in vitro immunogenicity of selected haptens.Immunogenic responses induced by 10-DL (also known as 140) and 10M-DL(also known as 140M) exhibit have a high quality index (Ab-titer/IC₅₀)(i.e., a high quality response (high titer and high affinity)).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION I. Terms and Abbreviations

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The practice of the techniquesdescribed herein may employ, unless otherwise indicated, conventionaltechniques and descriptions of organic chemistry, polymer technology,biochemistry, and sequencing technology, which are within the skill ofthose who practice in the art.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. As used herein, the term “substantially” as in, for example,the phrase “substantially all peptides of an array,” refers to at least90%, preferably at least 95%, more preferably at least 99%, and mostpreferably at least 99.9%, of the peptides of an array. Other uses ofthe term “substantially” involve an analogous definition.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

II. Overview

The compositions and methods provided herein are based, at least inpart, on the discovery of improved methods for designing nicotinehaptens. In particular, the inventors identified valuable parameters toguide rational hapten linker design and developed hapten-proteinconjugates having improved immunogenicity and vaccines that elicithigh-quality anti-nicotine (anti-Nic) antibody responses. Thus, thepresent invention overcomes the problems associated with known methodsof hapten design by providing methods for designing conjugate nicotinehaptens that, upon vaccination, specifically elicit antibodies havinghigh binding affinity to free nicotine and capable of achievingtherapeutically effective serum antibody titers, but having minimalaffinity for hapten linkers or carrier proteins.

III. Methods

Accordingly, in a first aspect, provided herein is a method of obtaininga nicotine hapten capable of eliciting an immune response specific tonicotine. The method comprises designing and selecting candidatenicotine hapten conjugates. As used herein, the term “hapten” refers toa small molecule that is substantially incapable of being immunogenic onits own but is immunogenic when conjugated to a higher molecular weightcarrier. As used herein, the term “hapten conjugate” refers to compoundformed by the union of a hapten and at least one other compound.Examples of hapten conjugates include, but are not limited to,hapten-antibody conjugates, hapten-polypeptide conjugates,hapten-linker-polypeptide conjugates, and labeled probes. Generally,conjugates are formed by joining, bonding, or otherwise linking onemolecule to another molecule to make a larger molecule. For example, ahapten can be covalently attached to another molecule such as a protein,antibody, nucleic acid, or lectin. In some cases, the protein is anavidin such as streptavidin. Streptavidin (SA) is a protein of bacterialorigin that specifically binds biotin to the substantial exclusion ofother small molecules that might be present in a biological sample.

Exemplary haptens for use according to the methods as provided hereininclude drugs, hormones, and toxins, but are not limited to thesespecific haptens. Any small molecule for which a vaccine would be usefulmay be used according to the methods provided herein. In exemplaryembodiments, the small molecule is a drug of addiction or abuse such as,for example, nicotine, cocaine, or heroin. In other cases, haptens canbe peptides, glycosylated peptides, metabolites, vitamins, hormones,prostaglandins, and toxins.

In a first aspect, provided herein is a method for obtaining a nicotinehapten capable of eliciting an immune response specific to nicotine. Themethod comprises the following steps: (a) estimating the length andrigidity of hapten linkers, according to their chemical structures.These haptens are ranked based on their rigidity, from high to low. Themethod further comprises (b) determining the accessibility of nicotineto an anti-nicotine antibody (e.g., monoclonal anti-nicotine antibody5F3) by competition ELISA in comparison to binding of the antibody tofree nicotine to derive an affinity index (ranging from 0.004 to >1),which is somewhat inversely correlated with the extent of the estimatedrigidity; (c) providing three-dimensional structural information of animmunogenic carrier; (d) selecting functional groups or small moleculefragments predicted to bind to free nicotine at an immunogenic carrierbinding site, wherein a functional group or small molecule fragment isselected if indicated to exhibit higher binding energy for free nicotinethan for the immunogenic carrier; and (e) linking the selectedfunctional group or small molecule fragment in a single compound.Preferably, the compound is a nicotine hapten that, when conjugated tothe immunogenic carrier, elicits production of anti-nicotine antibodieshaving an affinity index greater than 0.004 and less than or equal to 1.More preferably, the affinity index is greater than 0.002 and less than1.

As used herein, the term “rigid” refers to a molecule's propensity toadopt a defined conformation in preference to a variety of competingconformations. Such rigidity can be imparted by a variety of molecularfeatures that provide a bias in favor of a local conformation about amolecular skeleton. The term “rigidity” also refers to the degree offlexibility of the molecule and includes the terms “flexible,”semi-rigid,” “rigid,” and all variations in between.

To select a chemical structure that structurally mimics a targetmolecule (e.g., nicotine), it is advantageous to analyzethree-dimensional structural information. Such analysis can involveperforming molecular modeling. Generally, molecular modeling techniquesare useful for de novo drug design. In some cases, molecular modeling iscomputer-aided molecular modeling. As used herein, “computer-aidedmolecular modeling” refers to computer program-assisted analysis ofproperties relevant to antigen-antibody recognition including, withoutlimitation, three-dimensional polypeptide conformations, electrostaticpotential isosurfaces, and steric and electrostatic fields. In somecases, molecular modeling involves the identification of functionalgroups or small molecule fragments that can interact with the bindingsurface of nicotine and exert a complement-inhibiting biological effect.Once such functional groups or fragments are identified, they are linkedinto a single compound using bridging components having suitable sizeand geometry to fit a binding site on nicotine and exert a biologicaleffect. Computer programs useful for designing geometrically appropriatelinks and bridges for functional groups are known in the art and arepreferred over manual techniques for use in the present invention. Forexample, a docking software program such as AutoDock4 and itsaccompanying graphical user interface, AutoDockTools, can be usedaccording to a method provided herein. AutoDock programs are availableat autodock.scripps.edu and at mgltools.scripps.edu on the World WideWeb.

Selection of functional groups can comprise performing analysis ofdocking conformations to determine one or more binding energy parametersbetween nicotine and a binding site of the immunogenic carrier. As usedherein, the terms “binding pocket” and “binding site” are usedinterchangeably and refer to a region of a molecule or molecularcomplex, that, as a result of its shape, favorably associates withanother chemical entity or compound.

In some cases, computational means are employed to analyzethree-dimensional structural information. For example, a softwareprogram operated by a computer having a non-transitory computer-readablestorage medium containing the program can be used to analyze thestructural information and characterize the association between nicotineand the binding pocket. In exemplary embodiments, such computer-aidedmolecular modeling utilizes three-dimensional macromolecular structuralinformation obtained from experimental methodologies such as X-raycrystallography and nuclear magnetic resonance (NMR) spectroscopy.Macromolecular structural information can be compiled in a database.Preferably, molecular modeling includes consideration of anyperturbations in the three-dimensional conformation of the hapten, aswell as concomitant changes in its electronic distribution, that canoccur when a small molecule such as nicotine is conjugated to a carrierprotein. Generally, small molecules that are generally too small toinduce an immune response can be conjugated to a carrier protein byusing a linker. However, introduction of a linker or spacer arm betweenthe hapten and carrier protein can disturb the hapten'sthree-dimensional conformation. Accordingly, it is preferable thatselection of hapten candidates comprise molecule modeling of candidateshaving an attached linker or spacer. By including the linker or spacerarm in computational modeling, it is possible to discern possiblyeffects on the hapten resulting from a spacer or linker or conjugationchemistry.

In further steps, methods provided herein comprise selecting one or morefunctional groups or small molecule fragments indicated to bind morestrongly to the target molecule (e.g., nicotine) than to a non-targetmolecule. Selecting can involve analysis of calculations, in some casesmade by a computer or person. Relevant calculations can include, withoutlimitation, calculating theoretical geometries and electronicdistributions, as well as theoretical pKa values for deprotonationenthalpy (DPE) and formation enthalpy (HO).

In preferred embodiments, haptens designed and obtained according tomethods provided herein have intermediate binding affinity, i.e., theiraffinity index smaller than 1, but larger than 0.02. Haptens having suchan affinity index are considered to be good hapten candidates as theyhave a balanced influence of the linker effect. As used herein, a haptenhaving an intermediate index is a hapten capable of eliciting productionof antibodies that compete with free nicotine molecules but that exhibitno cross-reactivity to non-active hapten components. Percentcross-reactivity is defined as (50% inhibition of control (IC₅₀) of thetarget analyte/IC₅₀ of another compound)×100. Preferably,cross-reactivity calculations are performed using IC₅₀ values with unitsof moles (e.g., nmol/mL⁻¹ or pmol/mL⁻¹). Exemplary nicotine haptens arerepresented in FIG. 1.

Preferably, anti-nicotine antibodies bind to the hapten and any desiredpharmacologically active metabolites. In some cases, affinity andcross-reactivity profiles of each hapten-elicited antibody is determinedusing any appropriate assay such as a radioimmunoassay. As used herein,“determining” means quantitatively analyzing for the amount of asubstance. Preferably, determining comprises analyzing the IC₅₀ value(meaning “50% inhibition of control”) for inhibition of free nicotinefor antibodies generated against the nicotine hapten.

In preferred embodiments, selecting a structure that structurally andelectronically mimics the target molecule further comprises performingan enzyme-linked immunosorbent assay (ELISA) to identify a hapten havingan intermediate index. For example, an ELISA can be performed withplates coated with nicotine hapten conjugated to different carriermolecules to avoid selecting carrier molecule-reactive antibodies. Insome cases, the ELISA is an indirect competitive ELISA.

Methods provided herein can further comprise synthesizing or obtainingthe compound; and evaluating the compound for its ability to competewith free nicotine for binding. To elicit an antibody response to ahapten, it typically is covalently bound to a higher molecular weightmolecule such as a carrier protein, and the complex will elicit theproduction of antibodies that recognize the hapten. In some cases, theinteraction between a hapten and second molecule is direct. In othercases, the interaction involves at least one other molecule, e.g., alinker, spacer, or coupling agent. As used herein, the terms “linker,”“spacer,” and “coupling agent” are interchangeable and refer to amolecule or group of atoms positioned between two moieties. A linker orspacer may be formed using a molecule that is differentiallyfunctionalized or activated with groups at either end to allow selectivesequential reaction with the hapten and the carrier, but the samereactive moiety may also be used at both ends. For example, ahapten-protein conjugate may include a linker between the hapten and thepolypeptide or peptide. Typically, linkers are bifunctional, meaningthat the linker includes a functional group at each end, where thefunctional groups are used to couple the linker to the two moieties. Thetwo functional groups may be the same (i.e., a homo-bifunctional linker)or different (i.e., a heterobifunctional linker). The groups selectedfor reaction with the hapten and the functional linking group to bebound to the carrier are determined by the type of functionality on thehapten and the carrier to which the hapten is to be bonded.

Any suitable carrier having immunogenic properties can be used, andconventional conditions for the coupling reaction can be employed.Immunogenic carriers are often proteins, but carriers can also include asugar or fat in mono- or polymer form. In exemplary embodiments, thecarrier molecule is streptavidin. The binding of the hapten to thecarrier protein is often covalent, but it can be ionic or be effectedthrough a chemical component bridging the hapten and the carrier. Undercertain conditions it is possible to crosslink the hapten and no carriersubstance is needed in order to make it immunogenic.

In some cases, the carrier protein is a member of a “specific bindingpair,” meaning two different molecules wherein one of the molecules,through chemical or physical means, specifically binds to the secondmolecule. In addition to antigen and antibody specific binding pairs,other specific binding pairs include, as examples without limitation,biotin and avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), and the like.

Any appropriate technique can be used to detect and measure the degreeof conjugation of a nicotine hapten to an immunogenic carrier. Forexample, conjugation of a nicotine hapten to a polypeptide carrier canbe estimated using a technique such as a protein assay or ultraviolet(UV) spectral analysis.

Any appropriate technique can be used to confirm the chemical structureof a nicotine hapten obtained according to the methods provided herein.For example, the chemical structure of a nicotine hapten can beconfirmed by mass spectrometry, ¹H-NMR spectrometry, or ¹³Cspectrometry.

In another aspect, provided herein is a method of eliciting an immuneresponse against a small molecule in a subject. The method comprises orconsists essentially of administering to a subject in need thereof acomposition comprises a nicotine hapten, thereby eliciting an immuneresponse in the subject. In some cases, nicotine antibody-producingcells are isolated from an immunized subject, immortalized, and screenedfor the production of monoclonal antibodies. These monoclonal antibodiesmay, if desired, be prepared recombinantly by isolating the nucleicacids encoding them from the antibody-producing cells and manipulatingthe nucleic acids for recombinant production. Accordingly, modifiedforms of the antibodies, such as single-chain or Fv antibodies may beproduced.

In a further aspect, provided herein is a method of producing antibodiesto a nicotine hapten conjugate. The method comprises administering to ananimal an effective amount of a nicotine hapten conjugate providedherein; isolating antibodies from sera of the animal; and recovering theisolated antibodies. Antibodies produced according to the method areanti-nicotine antibodies having specificity for the nicotine portion ofthe conjugate. In some cases, the antibody is a polyclonal antibody.

IV. Compositions

In another aspect, provided herein is an immunogenic conjugatecomprising a nicotine hapten linked to a carrier molecule, where theconjugate elicits an immune response in a subject. Where the hapten is adrug of abuse or addiction such as nicotine, therapeutic compositionscomprising hapten-carrier conjugates as described herein areparticularly useful in the treatment of nicotine addiction. Suchtherapeutic compositions can be suitable for co-therapy with otherconventional drugs. Passive immunization using antibodies raised againstconjugates of the instant invention is also contemplated.

In a further aspect, provided herein are compositions comprisingnicotine hapten conjugates as described herein and at least one othercomponent, preferably at least one excipient or carrier and, mostpreferably, at least one pharmaceutically acceptable excipient orcarrier. In exemplary embodiments, a composition of the inventioncomprises an immunogenic nicotine hapten conjugate and apharmaceutically acceptable carrier.

The conjugates and compositions provided herein are useful for inducingimmune responses against nicotine haptens and include nicotine vaccines.Such an anti-nicotine immune response can be utilized to generateantibodies, including those suitable for therapeutic, prophylactic, anddiagnostic purposes. An anti-nicotine immune response can be useful toprevent or treat addiction to drugs of abuse and the resultant diseasesassociated with drug addiction.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal. Typically, thevaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the vaccine is able to provokean immune response including, but not limited to, the production ofantibodies and/or cytokines and/or the activation of cytotoxic T cells,antigen presenting cells, helper T cells, dendritic cells and/or othercellular responses. Optionally, the vaccine of the present inventionadditionally includes an adjuvant which can be present in either a minoror major proportion relative to the compound of the present invention.Without being bound by any particular mechanism, it is believed that theuse of a nicotine vaccine can induce a “memory” immune response uponexposure to nicotine and provide long-term anti-nicotine immunity.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may specifically bind the products of specific humoral orcellular immunity, such as an antibody molecule or T-cell receptor.Antigens can be any type of molecule including, for example, haptens,simple intermediary metabolites, sugars (e.g., oligosaccharides),lipids, and hormones as well as macromolecules such as complexcarbohydrates (e.g., polysaccharides), phospholipids, nucleic acids andproteins. As used herein, the term “epitope” refers to an antigenicdeterminant, meaning a particular chemical group or contiguous ornon-contiguous peptide sequence on a molecule that elicits a specificimmune response (“antigenic”). An antibody binds a particular antigenicepitope.

The term “antibody” is used herein in the broadest sense andspecifically encompasses at least monoclonal antibodies, polyclonalantibodies, multi-specific antibodies (e.g., bispecific antibodies),chimeric antibodies, humanized antibodies, human antibodies, andantibody fragments. An antibody is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes.

In some cases, a vaccine provided herein is administered with anadjuvant. As used herein, the term “adjuvant” refers to a compound ormixture of compounds that are added to or administered in conjunctionwith a vaccine in order to enhance antibody production efficacy or tohelp generate a specific class of antibodies as for example IgMimmunoglobulins or antibodies able to bind complement. Substancessuitable for use as an adjuvant include, without limitation, mineraloils, derivatives of aluminum, pathogen-associated molecular patterns(PAMPs) (e.g., lipopolysaccharide (LPS), porins, bacterial lipoproteinsand lipopeptides, peptidoglycan, lipoteichoic acids, mannose-richglycans, flagellin, bacterial and viral genomes, mycolic acid, andlipoarabinomannan), pattern-recognition receptors (PRRs), anddanger-associated molecular patterns (DAMPs) such as microbial DNA andRNA or heat-shock proteins. Vaccines provided herein can be administeredwith or without an adjuvant.

Any appropriate mode of vaccine administration can be used. In somecases, for example, a vaccine described herein is intended forparenteral, topical, oral, or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously (“i.v.”), subcutaneously (“s.c.”), intradermally, orintramuscularly, however other modes of administration may be devised.Thus, provided herein are compositions for parenteral administrationthat comprise a solution of the nicotine hapten conjugates describedabove dissolved or suspended in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of pharmaceutically acceptableaqueous carriers may be used including, without limitation, water,buffered water, saline, glycine hyaluronic acid, and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain as pharmaceuticallyacceptable carriers, substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

Nicotine hapten conjugates described herein are suitable forprophylactic uses as vaccines. For such uses, vaccines can be providedas a single administration or in two or more administrations orboosters.

Although the embodiments are described in considerable detail withreference to certain methods and materials, one skilled in the art willappreciate that the disclosure herein can be practiced by other than thedescribed embodiments, which have been presented for purposes ofillustration and not of limitation. Therefore, the scope of the appendedclaims should not be limited to the description of the embodimentscontained herein.

Examples Example 1—Rationale for Using Nic-Streptavidin Platform forHapten Design

To rationally design the nicotine vaccines, we selected streptavidin(SA) as a protein carrier, owing to the following properties: 1)well-characterized crystal structure and known residues for nicotineconjugation, which allows both molecular modeling and experimentalassessment of nicotine-streptavidin conjugates for examining theiraccessibility to nicotine-specific antibodies; 2) highly immunogenicnature; and 3) feasibility to make antigen-adjuvant complexes forenhanced immunity. With limited number of lysine residues forconjugation, the neighboring effect of conjugation sites can be modeledand assessed. On the other hand, also attributing to the same feature,the linker in the context of few conjugation sites per protein sites mayconstitute strong immunogenic epitopes to deter the immune reactionsthat are specific to free nicotine. Nevertheless, appropriateNic-haptens, e.g., those with minimal linker effects upon conjugation toSA, could focus the immunity solely toward nicotine, which is theultimate objective of this study. In our previous study, we categorizedhaptens according to their linker effect, which was calculated fromtheir interactions with monoclonal anti-Nic antibody (5F3) andnormalized against the binding activity of 5F3 to free nicotine.

One possibility is that a strong linker effect may constitute an epitopeindependent of the Nic moiety and interfere with immunity specific tothe Nic moiety. Another possibility is that some linker-mediated effectsmay also exert a positive influence in the interaction betweenNic-specific B cells and the Nic moiety, such as recruiting or promotingNic-specific B cells to engage with Nic moiety.

Nicotine haptens of different lengths and chemical properties areexpected to be highly variable in terms of conjugation efficiency,surface display, and linker effects on neighboring Nic moieties. Thus,it would be difficult or impossible to screen all variables in an animalmodel to identify those specific to Nic moiety. Instead, we combined thecomputational modeling approach with accessibility analysis based oncompetition ELISA to screen nicotine-streptavidin conjugates. Theidentified candidates were further tested for their immunogenicity andfunctionality.

To examine how newly synthesized nicotine interacts withnicotine-specific B cells, we used a nicotine-specific monoclonalantibody (5F3, with binding K_(d) at 66 nM) to test its binding to thesehaptens. We argued that if the haptens displayed worse binding activityto the antibody than free nicotine, they would have structuraldisadvantage in competing for the interaction with nicotine-specific Bcells, therefore these haptens would not be good vaccine candidates. Onthe other hand, many nicotine-specific antibodies bind better to theircognate nicotine haptens than free nicotine, in part attributed to theinteraction of Fab conjugated to the linker region of the hapten.Although the linker effect may help secure the interaction withnicotine-specific B cells the dependence on the linker effect forattracting these B cells may also result in the recruitment of B cellswith low intrinsic affinity in binding to free nicotine. Thus, weexpected that the ideal haptens should exhibit antibody-binding affinityat the level close to the one seen with free nicotine. Thus, usingcompetition ELISAs, we determined the binding activity of varioushaptens to 5F3 (expressed as IC_(50 HP)), and normalized by the IC₅₀ offree nicotine to express as relative affinity index(IC_(50HP)/IC_(50Nic)). The higher the number reflects the lower therelative affinity. As summarized in FIG. 12, several haptens have therelative affinity larger than 1, indicating their worse binding to 5F3than the one seen with free nicotine. In contrast, hapten 52, 45 and 138show very high relative binding affinity (with the index less than0.01). We suspect that the binding of these haptens to 5F3 is likelydriven by the linker effect, which ultimately may compromise theselection for high-affinity nicotine-specific B cells. Thus, based onthis analysis, haptens with intermediate binding affinity, i.e., theiraffinity index smaller than 1, but larger than 0.02, was considered asgood hapten candidate as they have a balanced influence of the linkereffect.

In order to assess the accessibility of free Nic moiety in the contextof SA, we conducted molecular modeling, which eventually provided someinsights about how well these nicotine haptens can be presented forantibody interactions. Given the well-characterized three-dimensionalstructure of SA, four lysine residues on the surface of SA are primarysites for Nic conjugation, i.e., K80, K121, K132 and K134. We attemptedto predict the binding affinity and conformation of different Nichaptens to SA using a rigid docking algorithm in the AutoDock4 program.In general, the binding conformation with the lowest energy isindicative of the better binding conformation of the ligand towards thereceptor protein that has stronger binding affinity through interactionwith ligands via neighboring amino acids. In the case of Nic haptens,the Nic moiety of the hapten should be stood out from the surface of theSA as much as possible in order to be available to interact with theanti-Nic antibody, and Nic-specific B cells. Here, we speculate that theconformation of Nic hapten having higher binding affinity to SA mightresult in reducing the availability of Nic moiety for antibodyinteraction. In other words, SA amino acids surrounding the Nic hapteninfluences the anti-Nic antibody interactions by making it leastavailable. Therefore, we predicted that a good Nic hapten which elicitsfocused immunogenicity to the free Nic moiety should exhibit highbinding energy.

We compared the binding energy of Nic haptens to four lysine positionsin SA and analyzed their docking conformations. The conformations andinteractions of different Nic haptens exhibited different behavior inthese four lysine regions of SA. The streptavidin-hapten conjugation isexpected to be between the reactive group at the linker end of the Nichapten and the lysine side chain of the streptavidin. The docking of Nichapten on to the streptavidin resulted in different conformations andorientations of Nic hapten near the exposed lysine residue along withthe presence of expected conjugatable conformation. This is obvious dueto the limitation that this docking algorithm cannot be able toprecisely form the expected covalent conjugation reaction between theNic hapten and streptavidin as is done experimentally. Rather thanmerely looking at the lowest energy conformation of Nic hapten, weanalyzed for the presence of Nic hapten conformations having the linkerend close to the lysine side chain with an interactable distance of 1.5to 2.5 Å which may resemble the expected conjugation. For some Nichaptens at some lysine positions, our docking analysis did not predictthese kind of expected conjugation which might be due to the influenceof neighboring amino acids interactions that are incompatible with theNic hapten at this kind of particular conformation. For thecomputational screening, selection and validation of Nic haptens, weutilized the binding energy parameter which reflect the affinity ofthese Nic haptens towards streptavidin. And for the comparison, weaveraged the binding energy of Nic hapten at all four lysine positionsfor each Nic hapten.

First, we compared the lowest binding energy conformation of Nic haptenstowards streptavidin irrespective of the presence of conformations withexpected conjugation. This comparison may reflect the influence ofneighboring amino acids of lysine in streptavidin to Nic haptens. TheNic haptens having lower binding energy may be worse than other Nichaptens because they interact strongly with streptavidin which mayaffect the antibody interaction. Second, we compared the binding energyof those which showed the expected conjugatable conformation of Nichapten on to streptavidin. The Nic haptens having higher binding energywith this conjugatable conformation are considered good since theysatisfy two expected criteria: one is having low binding affinity due tothe higher binding energy and secondly, possessing the conformationcompatible for conjugation. Since, some Nic haptens failed to show anyexpected conjugatable conformation at one or more lysine positions, theyare also regarded as worse than those having expected conjugatableconformation at all four lysine positions.

Using competition ELISA, we examined the binding activity ofNic-specific mAb (5F3) to conjugated nicotine, in comparison to the oneinteracting with haptens alone. Based on the number of lysine residuesper SA monomer, 16 lysine residues displayed on the surface of SAparticipate in conjugation to nicotine haptens. Thus, the fold ofincrease in binding activity from haptens to hapten-SA conjugates shouldonly reach to 16. However, the competition ELISA data shows that theestimated affinity is much higher than 16, ranging from 30 to >500.These data suggest that the inclusion of streptavidin, especially localresidues surrounding conjugated nicotine haptens, increases the overallbinding of hapten-SA conjugates to 5F3.

If the interaction between 5F3 and Nic-SA somehow fixes the nicotinemoiety in a certain conformation that resembles free nicotine in aqueousphase, the interaction should increase the efficacy of anti-Nic antibodyresponses. However, it is possible that the interactions produces a“linker effect” in which the linker is highly immunogenic and cause anincreased antibody response toward the linker rather than free nicotine.

Considering these two possible scenarios, we reasoned that the effect ofSA on the interaction between nicotine haptens and 5F3 should be at anintermediate level, where the SA linkage increases anti-Nic antibodyresponses with minimal linker-specific immunogenicity. Thus,computer-assisted modeling was combined with competition ELISA toidentify haptens having intermediate indexes. We selected 140-hapten(“VA-II-140”) as the best candidate. See FIG. 1. To validate ourselection, 140-hapten along with the previously described nicotinehapten (1′Nic), 137 (which binds poorly to mAb), and 138 (which has astrong linker effect) were conjugated to SA for immunogenicity assays.

Balb/c mice were immunized with nicotine-SA conjugates three times, andserum was collected from each mouse after the 2nd and 3rd immunizations.As shown in FIG. 10, after three rounds of immunization, the level ofanti-nicotine antibodies were significantly increased in mice immunizedwith 138-SA, 140-SA, whereas the anti-nicotine antibodies levels wererather low in mice immunized with 1′-Nic-SA and 137-SA. We also analyzedthe relative binding affinity of elicited anti-nicotine antibodies forboth free nicotine and their cognate haptens. Interestingly, for theantibody made in 138-immunized mice, although IC₅₀HP are relatively low,the IC₅₀ to free nicotine is quite high, indicating their low affinity.

Small scale analysis was repeated for the same haptens. Hapten 140 and140M (also referred to as 10 and 10M in, for example, FIG. 14) againexhibited induction of high quality antibody responses as determined byhigh titer and high affinity (low IC₅₀).

Materials and Methods

Products were concentrated by rotary evaporation (vacuum pump, ca.7.5-25 Torr). Analytical thin-layer chromatography was performed usingglass plates pre-coated with silica gel (0.25 mm, 60 Å pore size,230-400 mesh, Silicycle) impregnated with a fluorescent indicator (254nm). TLC plates were visualized by exposure to ultraviolet light (UV).Flash-column chromatography was performed employing silica gel (60 Åpore size, 40-63 μm, standard grade, Silicycle).

¹H NMR spectra was recorded on a Varian INOVA 400 (400 MHz) spectrometerat 25° C. Proton chemical shifts are expressed in parts per million(ppm, δ scale) and are referenced to residual protium in the NMR solvent(chloroform-d). Data are represented as follows: chemical shift,multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,brs=broad singlet), coupling constant (J) in Hertz (Hz) and integration.

Ethyl 5-bromonicotinate (1)

To an ice-cool solution of 5-bromonicotinic acid (15 g, 75 mmol) inethanol (250 mL) was added concentrated sulfuric acid (4 mL) slowly dropwise and refluxed under argon for 18 h. Ethanol was removed underreduced pressure and resulting white residue was dissolved in water. Theaqueous solution was made basic to pH 8 with sat. sodium bicarbonate andextracted with ether. The organic layer was washed with brine, driedover anhydrous MgSO₄, and concentrated under diminished pressureafforded 1 as a pale yellow solid: yield 15.20 g (89%); ¹H NMR (400 MHz,CDCl₃) δ 1.39 (t, 3H, J=7.2 Hz), 4.40 (q, 2H, J=7.6, 14.8 Hz), 8.40 (t,1H, J=2.0 Hz), 8.81 (d, 1H, J=2.4 Hz) and 9.10 (d, 1H, J=1.6 Hz); ¹³CNMR (100 MHz, CDCl₃) δ 14.3, 62.0, 120.7, 127.6, 139.5, 149.0, 154.5 and164.1.

3-Bromo-5-(4, 5-dihydro-3H-pyrrol-2-yl)-pyridine (2)

Sodium hydride (3.44 g, 86 mmol, 60% dispersion in oil) in a three-neckflask was washed with three 20 mL portions of hexane. The flask wasfitted with a reflux condenser, flushed with argon, and charged with THF(70 mL). A solution of 1 (10 g, 66.1 mmol) and 1-vinyl-2-pyrrolidinone(7.89 g, 71 mmol) in THF (15 mL) was added in one portion. The mixturewas stirred and refluxed for 1 h and then cooled to room temperature. Asolution of concentrated HCl (12 mL) in water (18 mL) was added, and theTHF was removed on a rotary evaporator. Additional concentrated HCl (18mL) and water (36 mL) were added, and the mixture was heated at refluxovernight. In an ice-cooled bath, the solution was made basic withconcentrated aqueous NaOH, which resulted in precipitation of the crudeproduct, and then it was extracted with CH₂Cl₂ (2×75 mL). The combinedCH₂Cl₂ layer was washed with water, brine, dried over anhydrous MgSO₄and concentrated under diminished pressure. The residue was purified bychromatography on a silica gel column (15×5 cm) eluting with 19:1CH₂Cl₂-acetone afforded 2 as a pale yellow solid: yield 6.87 g (70%); ¹HNMR (400 MHz, CDCl₃) δ 2.00-2.08 (m, 2H), 2.86-2.92 (m, 2H), 4.02-4.08(m, 2H), 8.31 (t, 1H, J=1.6 Hz), 8.67 (d, 1H, J=2.0 Hz) and 8.83 (d, 1H,J=1.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 22.6, 34.9, 61.8, 121.0, 131.7,137.2, 147.1, 152.2 and 169.9.

3-Bromo-5-(2-pyrrolidinyl)-pyridine (3)

A solution of compound 2 (2.0 g, 8.8 mmol) in 80 mL of 80:20methanol/acetic acid was cooled at −40° C. with dry ice-acetonitrilebath. To this reaction mixture sodium borohydride (747 mg, 19.75 mmol)was added portion wise over 10 min with vigorous stirring. During thecourse of the addition, the temperature rose to −20° C. After warming toroom temperature, most of the solvent was removed with a rotaryevaporator. Water (200 mL) was added and the solution was made basicwith NaOH and extracted with DCM (2×90 mL). The combined extracts werewashed with brine, dried over K₂CO₃, and evaporated. The residue waspurified by flash chromatography on a silica gel column (10×4 cm)eluting with 1:1 ethyl acetate-methanol afforded racemic 3 as a yellowoil: yield 1.8 g (90%); ¹H NMR (400 MHz, CDCl₃) δ 1.56-1.66 (m, 1H),1.81-1.95 (m, 3H), 2.16-2.25 (m, 1H), 3.00-3.06 (m, 1H), 3.11-3.17 (m,1H), 4.15 (t, 1H, J=7.6 Hz), 7.88 (t, 1H, J=1.6 Hz), 8.46 (d, 1H, J=2.0Hz) and 8.50 (d, 1H, J=2.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 25.6, 34.7,47.1, 59.3, 120.9, 136.8, 142.9, 146.8 and 149.2.

Resolution of Racemic 5-Bromonornicotine

To a solution compound 3 (2.33 g, 10.3 mmol) in ethyl acetate (16 mL)was added a solution of (−)-MTPA (1.2 g, 5.15 mmol) in ethyl acetate (4mL) with stirring. The mixture was allowed to stand at room temperaturefor 15 min, after which time the crystalline product was collected byfiltration to give (R)-isomer enriched crystals. Threerecrystallizations from boiling acetonitrile yielded 1.28 g (54%) ofcolorless needles [(R)-5-bromonornicotine (−)-MTPA salt]. The filtratewas extracted with 1 N sulfuric acid (2×15 mL). The acid extracts werecombined, washed with ether, made basic with NaOH, and extracted withDCM. The organic layer was evaporated to give an (5)-isomer enrichedoil. The oil was dissolved in ethyl acetate (9 mL) and treated with asolution of (+)-MTPA (1.2 g, 5.15 mmol) in ethyl acetate (4 mL) withstirring. After the solution was left standing for 15 min, thecrystallized product was collected by filtration. Threerecrystallizations from boiling acetonitrile yielded 1.20 g (51%) ofcolorless needles [(S)-5-bromonornicotine (+)-MTPA salt].

(S)-Nornicotine (5)

A suspension of (S)-5-bromonornicotine (+)-MTPA salt (500 mg, 1.08 mmol)in ether (80 mL) was vigorously shaken with 1 M KOH (30 mL) in aseparating funnel. The ether layer was washed with 1 M KOH (30 mL),dried over anhydrous K₂CO₃, and evaporated. The residual oil wasdissolved in ethanol (30 mL) containing Et₃N (0.6 mL) and hydrogen gaswas babbled through the reaction mixture with 10% Pd/C (200 mg). After 1h, the mixture was filtered through celite and the filter cake washedwith ethanol. The filtrate was poured into 1 M K₂CO₃ (90 mL) which wasthen extracted with CH₂Cl₂ (2×100 mL). After washing with brine (50 mL),the combined extracts were dried over anhydrous K₂CO₃ and concentratedunder diminished pressure. The residue was purified by flashchromatography on a silica gel column (10×1 cm) eluting with 7:1CH₂Cl₂-methanol afforded 5 as a pale yellow oil; yield 85 mg (53%);¹HNMR (400 MHz, CDCl₃) δ 1.56-1.66 (m, 1H), 1.78-1.91 (m, 2H), 1.99(brs, 1H), 2.11-2.20 (m, 1H), 2.96-3.02 (m, 1H), 3.11-3.17 (m, 1H), 4.10(t, 1H, J=7.6 Hz), 7.17-7.20 (m, 1H), 7.64-7.67 (m, 1H), 8.42 (dd, 1H,J=1.6, 4.8 Hz) and 8.54 (d, 1H, J=2.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ25.5, 34.4, 47.0, 60.1, 123.4, 134.1, 140.3, 148.3 and 148.6;MALDI-FTMS: 149 (MW).

(S)-Methyl 4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butanoate (6a)

A solution of methyl 4-iodobutanoate (227 mg, 1.21 mmol) in acetonitrile(0.4 mL) was added to a stirred solution of 5 (150 mg, 1.01 mmol) anddiisopropylethylamine (0.53 mL, 3.03 mmol) in acetonitrile (0.8 mL) atroom temperature. After 18 h, reaction mixture was concentrated underdiminished pressure, residue was directly purified by flashchromatography on a silica gel column eluting (15×2 cm) with 5% methanolin CH₂Cl₂ afforded 6a as a pale yellow oil: yield 196 mg (78%); silicagel TLC R_(f) 0.35 (5% methanol in CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ1.55-1.62 (m, 1H), 1.63-1.89 (m, 4H), 2.04-2.19 (m, 4H), 2.25-2.35 (m,1H), 2.38-2.43 (m, 1H), 3.19-3.30 (m, 2H), 3.52 (s, 3H), 7.18 (dd, 1H,J=4.8, 7.6 Hz), 7.62 (d, 1H, J=8.0 Hz), 8.42 (s, 1H) and 8.47 (s, 1H);¹³C NMR (100 MHz, CDCl₃) δ 22.7, 23.9, 31.7, 35.2, 51.4, 53.1, 53.2,67.5, 123.5, 134.9, 139.6, 148.5, 149.5 and 174.0; Mass spectrum(APCI+), m/z 249.1606 (M+H)⁺ (C₁₄H₂₁N₂O₂ requires m/z 249.1603).

(S)-Methyl 6-(2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoate (6b)

A solution of methyl 6-iodohexanoate (142 mg, 0.56 mmol) in acetonitrile(0.3 mL) was added to a stirred solution of 6 (75 mg, 0.51 mmol) anddiisopropylethylamine (0.26 mL, 1.53 mmol) in acetonitrile (0.8 mL) atroom temperature. After 18 h, reaction mixture was concentrated underdiminished pressure, residue was directly purified by flashchromatography on a silica gel column (15×2 cm) eluting with 3% methanolin CH₂Cl₂ afforded 6b as a pale yellow oil: yield 106 mg, (75%); silicagel TLC R_(f) 0.35 (5% methanol in CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ1.18-1.36 (m, 2H), 1.38-1.46 (m, 2H), 1.50-1.58 (m, 2H), 1.60-1.69 (m,1H), 1.78-1.88 (m, 1H), 1.89-2.00 (m, 1H), 2.02-2.09 (m, 1H), 2.14-2.27(m, 4H), 2.42-2.49 (m, 1H), 3.24 (t, 1H, J=8.0 Hz), 3.30-3.35 (m, 1H),3.64 (s, 3H), 7.22-7.25 (dd, 1H, J=4.8, 8.0 Hz), 7.67-7.70 (m, 1H), 8.48(d, 1H, J=3.6 Hz) and 8.54 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.6,24.8, 26.9, 28.4, 34.0, 35.2, 51.4, 53.6, 54.2, 67.7, 123.5, 134.9,139.7, 148.5, 149.5 and 174.1; Mass spectrum (APCI+), m/z 277.1925(M+H)⁺ (C₁₆H₂₅N₂O₂ requires m/z 277.1916).

(S)-4-(2-(Pyridin-3-yl)pyrrolidin-1-yl)butanoic acid (7a)

A solution of 6a (63 mg, 0.25 mmol) in methanol (1 mL) was added aq NaOH(30 mg, 0.76 mmol) with stirring at room temperature. After 18 h, thereaction mixture was evaporated, dissolved in 2 mL of acetone and the pHwas adjusted to 7 by using acetic acid. Acetone was evaporated and thecrude product was directly purified by flash chromatography on a silicagel column (10×2 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded 7a asa pale yellow oil: yield 38 mg (64%); silica gel TLC R_(f) 0.3 (1:1.5methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.70-1.80 (m, 3H), 1.82-2.08(m, 2H), 2.17-2.29 (m, 2H), 2.30-2.42 (m, 2H), 2.54-2.61 (m, 1H),3.44-3.51 (m, 3H), 7.32 (d, 1H, J=5.6 Hz), 7.84 (d, 1H, J=8.0 Hz), 8.50(s, 1H), 8.55 (s, 1H) and 11.30 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ22.4, 23.2, 33.8, 34.3, 53.3, 53.6, 67.7, 124.1, 135.9, 137.7, 148.3,148.9 and 176.6; Mass spectrum (APCI+), m/z 235.1452 (M+H)⁺ (C₁₃H₁₉N₂O₂requires m/z 235.1447).

(S)-6-(2-(Pyridin-3-yl)pyrrolidin-1-yl)hexanoic acid (7b)

A solution of 6b (100 mg, 0.36 mmol) in methanol (0.6 mL) was added aqNaOH (44 mg, 1.09 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, dissolved in 1 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×2 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded7b as a pale yellow oil: yield 76 mg (80%); silica gel TLC R_(f) 0.25(1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.18-1.36 (m, 2H),1.41-1.44 (m, 2H), 1.46-1.59 (m, 2H), 1.67-1.77 (m, 1H), 1.79-1.88 (m,1H), 1.91-2.02 (m, 1H), 2.08-2.21 (m, 2H), 2.23-2.31 (m, 3H), 2.44-2.51(m, 1H), 7.29 (d, 1H, J=7.2 Hz), 7.80 (d, 1H, J=7.6 Hz), 8.49 (d, 1H),8.55 (s, 1H) and 13.51 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.5, 25.0,27.0, 28.0, 34.7, 34.8, 53.3, 54.1, 67.6, 123.9, 136.0, 139.2, 147.6,148.5 and 177.6; Mass spectrum (APCI+), m/z 263.1761 (M+H)⁺ (C₁₅H₂₃N₂O₂requires m/z 263.1760).

(S,E)-Methyl 4-(2-(pyridin-3-yl)pyrrolidin-1-yl)but-2-enoate (8)

A solution of (E)-methyl 4-iodobut-2-enoate (134 mg, 0.59 mmol) inacetonitrile (0.3 mL) was added to a stirred solution of 5 (80 mg, 0.54mmol) and diisopropylethylamine (0.23 mL, 1.35 mmol) in acetonitrile(0.7 mL) at room temperature. After 18 h, reaction mixture wasconcentrated under diminished pressure, residue was directly purified byflash chromatography on a silica gel column (15×2 cm) eluting with 1:30methanol-CH₂Cl₂ afforded 8 as a pale yellow oil (97 mg, 73%); silica gelTLC R_(f) 0.45 (1:19 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ1.57-1.66 (m, 1H), 1.73-1.80 (m, 1H), 1.84-1.90 (m, 1H), 2.01-2.16 (m,2H), 2.17-2.24 (m, 1H), 2.99 (dd, 1H, J=6.8, 16.8 Hz), 3.64 (s, 3H),5.90 (d, 1H, J=15.6 Hz), 6.79-6.86 (m, 1H), 7.17 (dd, 1H, J=4.8, 8.0Hz), 7.62-7.65 (m, 1H), 8.41 (d, 1H, J=4.4 Hz) and 8.47 (d, J=2.0 Hz);¹³C NMR (100 MHz, CDCl₃) δ 22.8, 35.1, 51.5, 53.8, 54.4, 66.79, 122.0,123.7, 134.8, 138.8, 146.2, 148.8, 149.5 and 166.7; Mass spectrum(FAB+), m/z 247.1453 (M+H)⁺ (C₁₄H₁₉N₂O₃ requires m/z 247.1447).

(S,E)-4-(2-(Pyridin-3-yl)pyrrolidin-1-yl)but-2-enoic acid (9)

A solution of 8 (95 mg, 0.39 mmol) in methanol (0.8 mL) was added aqNaOH (47 mg, 1.16 mmol) with stirring at room temperature. After 15 h,the reaction mixture was evaporated, dissolved in acetone (2 mL) and thepH was adjusted to 7 by using acetic acid. Acetone was evaporated andthe crude product was directly purified by flash chromatography on asilica gel column (10×2 cm) eluting with 1:1.5 methanol-CH₂Cl₂ afforded9 as a pale yellow oil (71 mg, 79%) as a pale yellow oil; silica gel TLCR_(f) 0.4 (1:1.5 methanol-CH₂Cl₂); 1.64-1.76 (m, 1H), 1.79-1.88 (m, 1H),1.90-2.02 (m, 1H), 2.18-2.26 (m, 1H), 2.29-2.36 (m, 1H), 2.93 (dd, 1H,J=6.4, 15.6 Hz), 3.33 (d, 1H, J=10.0 Hz), 3.48 (t, 1H, J=7.2 Hz), 6.00(d, 1H, J=15.6 Hz), 6.88-6.95 (m, 1H), 7.32 (dd, 1H, J=4.8, 7.6 Hz),7.82 (d, 1H, J=8.0 Hz), 8.51 (d, 1H, J=4.0 Hz), 8.58 (s, 1H) and 12.30(brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.8, 35.0, 53.8, 54.4, 66.7,123.6, 124.2, 136.1, 139.3, 145.8, 147.7, 148.4 and 169.9; Mass spectrum(FAB+), m/z 233.1286 (M+H)⁺ (C₁₃H₁₇N₂O₂ requires m/z 233.1290).

(1r,4r)-Methyl 4-((tert-butoxycarbonyl)amino)cyclohexanecarboxylate (11)

To a stirred solution of(1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexanecarboxylic acid (10,150 mg, 0.617 mmol) in dry CH₂Cl₂ (5 mL) at 0° C. was added Et₃N (0.17mL, 1.234 mmol), after few minutes isopropylchloroformate (84 μL, 0.648mmol) was added. After 1 h stirring at 0° C., excess amount of methanol(0.5 mL) was added to reaction mixture and allowed to warm to roomtemperature. After 15 hours, reaction mixture was concentrated underdiminished pressure, residue was dissolved in CH₂Cl₂ (25 mL) and washedwith sat. NaHCO₃ solution, brine, dried over anhydrous MgSO₄ andconcentrated. The residue was purified by chromatography on silica gelcolumn (15×2 cm) eluting with 4:1 hexane-ethyl acetate afforded 11 as awhite solid: yield 147 mg (92%); silica gel TLC R_(f) 0.6 (4:1hexane-ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 1.03-1.13 (m, 2H), 1.40(s, 9H), 1.41-1.54 (m, 2H), 1.95-2.05 (m, 4H), 2.15-2.22 (m, 1H), 3.37(brs, 1H), 3.63 (s, 3H) and 4.42 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ27.9, 28.5, 32.6, 42.4, 49.1, 51.7, 79.3, 155.2 and 175.9;

(1r,4r)-Methyl 4-aminocyclohexanecarboxylate (12)

To an ice-cool solution of 11 (140 mg, 0.54 mmol) in dry CH₂Cl₂ (2 mL)was added trifluoroacetic acid (0.62 mL, 8.17 mmol) with stirring. After18 h at room temperature, reaction mixture was evaporated with toluene(1 mL×3 times) to give 12, which was directly used for the next reactionwithout any further purification.

(1s,4s)-Methyl 4-aminocyclohexanecarboxylate hydrochloride (14)

To a solution of (1s,4s)-4-aminocyclohexanecarboxylic acid (13, 50 mg,0.35 mmol) in methanol (2 mL) at 0° C. was added thionyl chloride (76μL, 1.05 mmol) drop wise. After addition the reaction mixture wasallowed to warm to room temperature and stirred for overnight. Reactionmixture was concentrated under diminished pressure yielded(1s,4s)-methyl 4-aminocyclohexanecarboxylate hydrochloride (14) as awhite solid, which was directly used in next reaction.

(1R,3S)-Methyl 3-aminocyclopentanecarboxylate hydrochloride (16)

To a stirred solution of (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one (15,100 mg, 0.91 mmol) in ethyl acetate (5 mL) was added 10% Pd/C (15 mg)and hydrogenated for 12 h by using hydrogen balloons at roomtemperature. Reaction mixture was filtered through celite bed, filtercake washed with ethyl acetate (3 mL). The combined organic layers wasconcentrated under reduced pressure afforded(1S,4R)-2-azabicyclo[2.2.1]heptan-3-one as a white solid, which wasdirectly used in the next reaction. To an ice-cool stirred solution of(1S,4R)-2-azabicyclo[2.2.1]heptan-3-one in methanol (3 mL) was addedthionylchloride (0.2 mL, 1.64 mmol). After 24 h at room temperature,reaction mixture was concentrated under diminished pressure afforded 16as a white solid: yield 109 mg (66%), which was directly used in thenext reaction; ¹H NMR (400 MHz, D₂O) δ 1.80-1.89 (m, 1H), 1.90-1.97 (m,1H), 1.99-2.06 (m, 1H), 2.09-2.13 (m, 1H), 2.14-2.21 (m, 1H), 2.42-2.50(m, 1H), 3.03-3.11 (m, 1H) and 3.76-3.82 (m, 4H (3+1)), ¹³C NMR (100MHz, D₂O) δ 27.4, 29.8, 33.6, 42.2, 51.4, 52.6 and 178.3.

tert-Butyl 4-(2-ethoxy-2-oxoethyl)piperazine-1-carboxylate (18)

To a stirred solution of N-Boc piperazine (17, 300 mg, 1.61 mmol) in dryDMF (2 mL) was added K₂CO₃ and ethyl iodoacetate (345 mg, 1.61 mmol) atroom temperature. After 15 h, the reaction mixture was diluted withwater (25 mL), extracted with ethyl acetate (2×30 mL). The combinedorganic phase was washed with brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was purified bychromatography on a silica gel column (10×2 cm) eluting with 1:1 ethylacetate-hexanes yielded 18 as a pale yellow oil: yield 320 mg (73%);silica gel TLC R_(f) 0.51 (1:1 ethyl acetate-hexanes); ¹H NMR (400 MHz,CDCl₃) δ 1.28 (t, 3H, J=7.2 Hz), 1.46 (s, 9H), 2.54 (t, 4H, J=4.8 Hz),3.23 (s, 2H), 3.48 (t, 4H, J=4.8 Hz), and 4.19 (q, 2H, J=7.2, 12.0 Hz);¹³C NMR (100 MHz, CDCl₃) δ 14.3, 28.5, 52.8, 59.5, 60.7, 79.7, 154.7 and170.2.

Allyl 1H-pyrrole-3-carboxylate (21)

To a solution of 1H-pyrrole-3-carboxylic acid (20, 160 mg, 1.44 mmol) inmethanol (5 mL) was added Cs₂CO₃ (470 mg, 1.44 mmol) and stirred for 10min at room temperature. The reaction mixture was concentrated underdiminished pressure and the residue was re-dissolved in DMF (20 mL). Tothis, allyl bromide (0.37 mL, 4.32 mmol) was added and stirred for 1 hat room temperature. The reaction mixture was poured in water (50 mL),extracted with ethyl acetate (2×20 mL). The combined organic phase waswashed with aq saturated NaHCO₃, water, brine and concentrated underdiminished pressure. The residue was purified by chromatography on asilica gel column (20×2 cm) eluting 1:4 ethyl acetate-hexanes afforded21 as a pale yellow liquid: yield 145 mg (67%); silica gel TLC R_(f)0.62 (1:4 ethyl acetate-hexanes); ¹H NMR (400 MHz, CDCl₃) δ 4.73-4.75(m, 2H), 5.22-5.25 (dd, 1H, J=1.2, 10.4 Hz), 5.34-5.38 (dd, 1H, J=1.6,17.2 Hz), 5.95-6.05 (m, 1H), 6.64-6.66 (dd, 1H, J=2.8, 4.0 Hz),6.73-6.75 (dd, 1H, J=2.4, 4.8 Hz), 7.42-7.44 (m, 1H) and 9.47 (brs, 1H);¹³C NMR (100 MHz, CDCl₃) δ 64.5, 109.6, 115.7, 117.6, 119.2, 124.0,132.7 and 165.4; Mass spectrum (FAB+), m/z 152.0707 (M+H)⁺ (C₈H₁₀NO₂requires m/z 152.0712).

(1S,4r)-Methyl4-(4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxyl-ate(22)

To a stirred solution of 7a (40 mg, 0.171 mmol) in dry DMF (0.7 mL) wasadded HBTU (71 mg, 0.188 mmol) at 0° C. After 10 min, a solution of 12and N-methylmorpholine (55 μL, 0.512 mmol) in dry DMF (0.4 mL) was addedat 0° C. After 18 h at room temperature, the reaction mixture waspartitioned between ethyl acetate (25 mL) and water (20 mL). The aqueouslayer was again extracted with ethyl acetate (20 mL), combined organiclayers was washed with water, brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was purified bychromatography on a silica gel column (10×1 cm), eluting with 5%methanol-CH₂Cl₂ afforded 22 as a pale yellow oil: yield 21 mg (33%);silica gel TLC R_(f) 0.31 (5% methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃)δ 1.45-1.57 (m, 2H), 1.68-1.81 (m, 3H), 1.88-2.01 (m, 6H), 2.06-2.36 (m,8H), 3.37-3.46 (m, 2H), 3.63-3.75 (m, 3+1 H), 5.67-5.80 (d, 1H),7.24-7.30 (m, 1H), 7.66-7.70 (m, 1H), 8.49 (d, 1H) and 8.56 (s, 1H); ¹³CNMR (100 MHz, CDCl₃) δ 22.6, 24.1, 27.5, 32.0, 34.6, 34.9, 42.3, 47.7,51.6, 53.4, 53.7, 67.8, 123.7, 135.1, 148.7, 149.4, 172.3 and 175.7;Mass spectrum (APCI+), m/z 374.2437 (M+H)⁺ (C₂₁H₃₂N₃O₃ requires m/z374.2444).

(1S,4r)-4-(4-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxylicacid (23)

A solution of 22 (21 mg, 0.056 mmol) in methanol (0.6 mL) was added aqNaOH (6 mg, 0.14 mmol) with stirring at room temperature. After 36 h,the reaction mixture was evaporated, dissolved in 1 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×1 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded23 as a pale yellow oil: yield 12 mg (59%); silica gel TLC R_(f) 0.25(1:1.5 methanol-CH₂Cl₂); ¹⁻H NMR (400 MHz, CDCl₃) δ 0.99-1.17 (m, 2H),1.50-1.60 (m, 2H), 1.66-1.81 (m, 3H), 1.83-1.93 (m, 2H), 1.94-2.06 (m,4H), 2.09-2.15 (m, 1H), 2.17-2.30 (m, 5H), 2.42-2.49 (m, 1H), 3.34-3.43(m, 2H), 3.62-3.77 (m, 1H), 6.11 (d, 1H, J=7.6 Hz), 7.31 (s, 1H), 7.73(d, 1H, J=7.6 Hz), 8.48 (s, 1H), 8.68 (s, 1H) and 11.30 (brs, 1H); ¹³CNMR (100 MHz, CDCl₃) δ 22.6, 23.9, 28.04, 32.08, 34.19, 34.94, 47.64,53.18, 53.55, 67.81, 123.71, 136.5, 139.2, 146.89, 148.09 and 172.16;Mass spectrum (APCI+), m/z 360.2282 (M+H)⁺ (C₂₀H₃₀N₃O₃ requires m/z360.2287).

(1R,4s)-methyl4-(4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxy-late(24)

To a stirred solution of 7a (38 mg, 0.162 mmol) in dry DMF (0.7 mL) wasadded HBTU (68 mg, 0.178 mmol) at 0° C. After 10 min, a solution of 14and N-methylmorpholine (53 μL, 0.487 mmol) in dry DMF (0.4 mL) was addedat 0° C. After 18 h at room temperature, reaction mixture waspartitioned between ethyl acetate (25 mL) and water (20 mL). The aqueouslayer was again extracted with ethyl acetate (20 mL), combined organiclayers was washed with water, brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was purified bychromatography on a silica gel column (15×1 cm), eluting with 5%methanol-CH₂C₁₂ afforded 24 as a pale yellow oil: yield (22 mg, 36%);silica gel TLC R_(f) 0.31 (5% methanol-CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃)δ 1.39-1.51 (m, 2H), 1.61-1.81 (m, 7H), 1.82-2.02 (m, 4H), 2.03-2.10 (m,1H), 2.14-2.39 (m, 4H), 2.44-2.52 (m, 2H), 3.32-3.45 (m, 2H), 3.68 (s,3H), 3.80-3.90 (m, 1H), 5.79-5.93 (brs, 1H), 7.25-7.31 (m, 1H), 7.69 (d,1H), 8.49 (s, 1H), and 8.55 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 22.49,24.20, 24.94, 29.03, 34.67, 39.69, 46.00, 51.57, 53.35, 53.77, 67.78,123.51, 135.01, 148.64, 149.36, 172.15 and 175.38; Mass spectrum(APCI+), m/z 374.2443 (M+H)⁺ (C₂₁H₃₂N₃O₃ requires m/z 374.2444).

(1R,4s)-4-(4-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxylicacid (25)

A solution of 24 (22 mg, 0.059 mmol) in methanol (0.6 mL) was added aqNaOH (12 mg, 0.295 mmol) with stirring at room temperature. After 36 h,the reaction mixture was evaporated, dissolved in 1 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×1 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded25 as a pale yellow oil: yield 11 mg (52%); silica gel TLC R_(f) 0.25(1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.32-1.39 (m, 1H),1.42-1.51 (m, 3H), 1.63-1.72 (m, 4H), 1.74-1.88 (m, 3H), 1.91-2.05 (m,3H), 2.11-2.29 (m, 5H), 2.40-2.54 (m, 2H), 5.62 (d, 1H), 7.24-7.35 (m,1H), 7.64 (d, 1H), 8.46 (s, 1H), 8.75 (s, 1H), 10.64-11.45 (brs, 1H);¹³C NMR (100 MHz, CDCl₃) δ 22.58, 23.76, 25.36, 29.49, 33.75, 34.85,46.11, 53.41, 53.63, 67.93, 123.58, 136.71, 146.55, 147.97, 171.91; Massspectrum (APCI+), m/z 360.2279 (M+H)⁺ (C₂₀H₃₀N₃O₃ requires m/z360.2287).

(1S,4r)-Methyl4-((E)-4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)but-2-enamido)cyclohexane-carboxylate(26)

To an ice-cool stirred solution of 9 (70 mg, 0.30 mmol) in dry DMF (0.9mL) was added HATU (126 mg, 0.33 mmol). After 10 min, a solution of 12and N-methylmorpholine (83 μL, 0.75 mmol) in dry DMF (0.4 mL) was addedat 0° C. After 18 h at room temperature, the reaction mixture wasdiluted with water (20 mL), extracted with ethyl acetate (2×20 mL). Thecombined organic phase was washed with brine, dried over anhydrous MgSO₄and concentrated under diminished pressure. The residue was purified byflash chromatography on a silica gel column (20×2 cm) eluting with 1:19methanol-CH₂Cl₂ afforded 26 as a pale yellow oil: yield 67 mg (60%);silica gel TLC R_(f) 0.3 (1:19 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃)δ 1.07-1.15 (m, 2H), 1.35-1.40 (m, 2H), 1.43-1.52 (m, 2H), 1.56-1.65 (m,1H), 1.74-1.97 (m, 6H), 2.00-2.26 (m, 3H), 2.77 (dd, 1H, J=7.2, 15.2Hz), 3.12-3.25 (m, 2H), 3.33 (t, 1H, J=8.0 Hz), 3.59 (s, 3H), 3.64-3.77(m, 1H), 5.67 (s, 1H, J=8.0 Hz), 5.83 (d, 1H, J=15.6 Hz), 6.61-6.69 (m,1H), 7.19 (dd, 1H, J=8.0, 12.4 Hz), 7.64 (d, 1H, J=8.0 Hz), 8.40 (s, 1H)and 8.45 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.8, 27.8, 32.1, 35.1,42.4, 47.8, 51.7, 53.8, 54.4, 66.7, 123.8, 125.2, 135.1, 139.1, 140.9,148.6, 149.4, 164.9 and 175.8; Mass spectrum (FAB+), m/z 372.2286 (M+H)⁺(C₂₁H₃₀N₃O₃ requires m/z 372.2287).

(1S,4r)-4-((E)-4-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)but-2-enamido)cyclohexanecarboxylicacid (27)

A solution of 26 (67 mg, 0.18 mmol) in methanol (0.7 mL) was added aqNaOH (22 mg, 0.54 mmol) with stirring at room temperature. After 36 h,the reaction mixture was concentrated under diminished pressure,dissolved in acetone (3 mL) and the pH was adjusted to 7 by using aceticacid. Acetone was evaporated and the crude product was directly purifiedby flash chromatography on a silica gel column eluting (10×2 cm) with1:1.5 methanol-CH₂Cl₂ afforded 27 as a colorless oil: yield 43 mg (67%);silica gel TLC R_(f) 0.25 (1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz,CD₃OD) δ 1.24 (q, 2H, J=11.6, 24.0 Hz), 1.42-1.53 (m, 2H), 1.65-1.75 (m,1H), 1.83-2.00 (m, 6H), 2.14-2.29 (m, 2H), 2.38 (q, 1H, J=8.4, 18.0 Hz),2.94 (dd, 1H, J=7.2, 16.4 Hz), 3.25-3.33 (m, 2H), 3.51 (t, 1H, J=8.4Hz), 3.59-3.67 (m, 1H), 6.02 (d, 1H, J=15.2 Hz), 6.61-6.69 (m, 1H), 7.39(t, 1H, J=7.6 Hz), 7.86 (d, 1H, J=8.0 Hz), 8.40 (d, 1H, J=3.6 Hz) and8.49 (s, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 23.7, 29.4, 32.9, 35.9, 44.1,55.0, 55.7, 62.2, 125.6, 126.9, 137.7, 140.7, 141.4, 149.2, 149.8, 167.2and 180.2; Mass spectrum (FAB+), m/z 358.2130 (M+H)⁺ (C₂₀H₂₈N₃O₃requires m/z 358.2131).

(1R,4s)-Methyl4-((E)-4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)but-2-enamido)cyclohexane-carboxylate(28)

To an ice-cool stirred solution of 9 (68 mg, 0.29 mmol) in dry DMF (0.7mL) was added HBTU (123 mg, 0.32 mmol). After 10 min, a solution of 14and N-methylmorpholine (97 μL, 0.88 mmol) in dry DMF (0.3 mL) was addedat 0° C. After 18 h at room temperature, the reaction mixture wasdiluted with water (20 mL), extracted with ethyl acetate (2×20 mL). Thecombined organic phase was washed with brine, dried over anhydrous MgSO₄and concentrated under diminished pressure. The residue was purified byflash chromatography on a silica gel column (20×2 cm), eluting with 1:19methanol-CH₂Cl₂ afforded 28 as a pale yellow oil: yield 52 mg (48%);silica gel TLC R_(f) 0.3 (1:19 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃)δ 1.51-1.57 (m, 2H), 1.60-1.75 (m, 4H), 1.77-1.94 (m, 4H), 2.13-2.29 (m,2H), 2.45-2.48 (m, 1H), 2.81 (dd, 1H, J=6.4, 15.6 Hz), 3.23-3.30 (m,2H), 3.37 (t, 1H, J=8.0 Hz), 3.59-3.66 (m, 4H (3+1)), 3.93-3.97 (m, 1H),5.60 (d, 1H, J=6.4 Hz), 5.88 (d, 1H, J=15.2 Hz), 6.66-6.73 (m, 1H), 7.23(d, 1H, J=11.2 Hz), 7.68 (d, 1H, J=7.6 Hz), 8.45 (s, 1H) and 8.51 (s,1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.8, 25.1, 25.2, 29.4, 29.7, 35.1,40.1, 46.0, 51.7, 53.8, 54.4, 66.8, 123.8, 125.2, 135.0, 139.0, 141.0,148.8, 149.5, 164.9, 175.7; Mass spectrum (FAB+), m/z 372.2283 (M+H)⁺(C₂₁H₃₀N₃O₃ requires m/z 372.2287).

(1R,4s)-4-((E)-4-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)but-2-enamido)cyclohexanecarboxylicacid (29)

A solution of 28 (50 mg, 0.14 mmol) in methanol (0.7 mL) was added aqNaOH (16 mg, 0.40 mmol) with stirring at room temperature. After 36 h,the reaction mixture was evaporated, dissolved in acetone (3 mL) and thepH was adjusted to 7 by using acetic acid. Acetone was evaporated andthe residue was directly purified by flash chromatography on a silicagel column (10×2 cm) eluting with 1:1.5 methanol-CH₂Cl₂ afforded 29 as acolorless oil: yield 32 mg (67%); silica gel TLC R_(f) 0.25 (1:1.5methanol-CH₂Cl₂); ¹H NMR (400 MHz, DMSO-d₆) δ 1.42-1.48 (m, 2H),1.52-1.61 (m, 5H), 1.77-1.84 (m, 4H), 2.13-2.27 (m, 2H), 2.37 (s, 1H),2.80 (dd, 1H, J=4.0, 16.0 Hz), 3.15-3.24 (m, 2H), 3.43 (t, 1H, J=7.6Hz), 3.73 (d, 1H, J=3.6 Hz), 6.08 (d, 1H, J=15.2 Hz), 6.48-6.55 (m, 1H),7.35 (dd, 1H, J=5.2, 8.0 Hz), 7.74 (d, 1H, J=8.0 Hz), 7.85 (d, 1H, J=7.2Hz), 8.45 (s, 1H), 8.52 (s, 1H) and 12.10 (brs, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 22.4, 24.6, 29.0, 34.7, 45.2, 48.6, 53.1, 53.9, 65.8, 123.7,125.7, 134.7, 138.9, 139.0, 148.4, 148.9, 163.8 and 176.1; Mass spectrum(FAB+), m/z 358.2124 (M+H)⁺ (C₂₀H₂₈N₃O₃ requires m/z 358.2131).

(S)-Methyl 4-(4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)benzoate(30)

To an ice-cool stirred solution of 7a (48 mg, 0.205 mmol) in dry DMF(0.7 mL) was added HBTU (94 mg, 0.246 mmol). After 10 min, a solution ofmethyl 4-aminobenzoate (37 mg, 0.246 mmol) and N-methylmorpholine (68μL, 0.615 mmol) in dry DMF (0.3 mL) was added at 0° C. After 18 h atroom temperature, the reaction mixture was diluted with water (20 mL),extracted with ethyl acetate (2×20 mL). The combined organic phase waswashed with brine, dried over anhydrous MgSO₄ and concentrated underdiminished pressure. The residue was purified by chromatography on asilica gel column (15×2 cm), eluting with 1:19 methanol-CH₂Cl₂ afforded30 as a pale yellow oil: yield 37 mg (49%); silica gel TLC R_(f) 0.3(1:19 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.71-1.95 (m, 5H),2.18-2.31 (m, 4H), 2.36-2.41 (m, 1H), 2.47-2.50 (m, 1H), 3.32-3.39 (m,2H), 3.87 (s, 3H), 7.16 (t, 1H, J=3.6 Hz), 7.58 (t, 2H, J=3.6 Hz), 7.67(s, 1H), 7.93-7.96 (m, 2H), 8.42 (s, 1H), 8.54 (s, 1H) and 8.78 (s, 1H);¹³C NMR (100 MHz, CDCl₃) δ 22.7, 24.0, 34.0, 35.7, 52.1, 53.5, 53.9,68.1, 118.8, 123.8, 125.2, 130.8, 135.4, 142.8, 148.6, 149.4, 166.8 and171.8; Mass spectrum (FAB+), m/z 368.1970 (M+H)⁺ (C₂₁H₂₆N₃O₃ requiresm/z 368.1974).

(S)-4-(4-(2-(Pyridin-3-yl)pyrrolidin-1-yl)butanamido)benzoic acid (31)

A solution of 30 (35 mg, 0.095 mmol) in methanol (0.5 mL) was added aqNaOH (12 mg, 0.286 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, residue was dissolved in acetone (3mL) and the pH was adjusted to 7 by using acetic acid. Acetone wasevaporated under diminished pressure and the residue was directlypurified by flash chromatography on a silica gel column (10×1 cm)eluting with 1:1.5 methanol-CH₂Cl₂ afforded 31 as a colorless oil (19mg, 57%); silica gel TLC R_(f) 0.22 (1:1.5 methanol-CH₂Cl₂); ¹H NMR (400MHz, DMSO-d₆) δ 1.54 (brs, 1H), 1.62-1.72 (m, 2H), 1.78-1.88 (m, 3H),2.08-2.17 (m, 2H), 2.20-2.27 (m, 2H), 2.31-2.41 (m, 2H), 2.47 (t, 1H,J=2.0 Hz), 7.21-7.25 (m, 1H), 7.65 (d, 2H, J=8.8 Hz), 7.69-7.77 (m, 1H),7.82 (d, 2H, J=9.2 Hz), 8.38 (d, 1H, J=4.0 Hz), 8.48 (s, 1H), 10.27 (s,1H) and 12.57 (brs, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 22.2, 23.7, 34.0,52.7, 66.8, 118.2, 120.6, 123.6, 124.9, 129.9, 130.3, 134.8, 143.4,148.4, 149.0, 167.0 and 171.5; Mass spectrum (FAB+), m/z 354.1819 (M+H)⁺(C₂₀H₂₄N₃O₃ requires m/z 354.1818).

(1R,3S)-Methyl3-(4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclopentane-carboxylate(32)

To an ice-cool stirred solution of 7a (38 mg, 0.162 mmol) in dry DMF(0.6 mL) was added HBTU (68 mg, 0.179 mmol). After 10 min, a solution of16 (35 mg, 0.195) and N-methylmorpholine (54 μL, 0.487 mmol) in dry DMF(0.3 mL) was added at 0° C. After 18 h at room temperature, the reactionmixture was diluted with water (20 mL), extracted with ethyl acetate(2×20 mL). The combined organic phase was washed with brine, dried overanhydrous MgSO₄ and concentrated under diminished pressure. The residuewas purified by flash chromatography on a silica gel column (20×1 cm),eluting with 1:9 methanol-CH₂Cl₂ afforded 32 as a pale yellow oil: yield27 mg (48%); silica gel TLC R_(f) 0.25 (1:9 methanol-CH₂Cl₂); ¹H NMR(400 MHz, CDCl₃) δ 1.42-1.49 (m, 1H), 1.54-1.61 (m, 1H), 1.66-1.88 (m,7H), 1.93-2.21 (m, 5H), 2.22-2.55 (m, 3H), 2.71-2.78 (m, 1H), 3.34-3.47(m, 2H, 3.57 (s, 3H), 4.12-4.18 (m, 1H), 6.40 (brs, 1H), 7.18 (t, 1H,J=2.8 Hz), 7.72 (s, 1H), 8.41 (d, 1H, J=4.0 Hz) and 8.45 (s, 1H); ¹³CNMR (100 MHz, CDCl₃) δ 22.4, 23.7, 28.3, 29.6, 32.9, 34.3, 36.0, 41.7,50.8, 52.0, 53.3, 53.5, 68.1, 123.7, 135.4, 149.1, 149.5, 172.1 and177.8; Mass spectrum (FAB+), m/z 360.2277 (M+H)⁺ (C₂₀H₃₀N₃O₃ requiresm/z 360.2287).

(1R,3S)-3-(4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclopentanecarboxylicacid (33)

A solution of 32 (26 mg, 0.072 mmol) in methanol (0.5 mL) was added aqNaOH (9 mg, 0.217 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, residue was dissolved in acetone (3mL) and the pH was adjusted to 7 by using acetic acid. Acetone wasevaporated under diminished pressure and the residue was directlypurified by flash chromatography on a silica gel column (10×1 cm)eluting with 1:1.5 methanol-CH₂Cl₂ afforded 33 as a colorless oil: yield19 mg (76%); silica gel TLC R_(f) 0.2 (1:1.5 methanol-CH₂Cl₂); ¹H NMR(400 MHz, CDCl₃) δ 1.42-1.51 (m, 1H), 1.61-1.91 (m, 9H), 2.00-2.27 (m,6H), 2.37-2.44 (m, 1H), 2.70-2.84 (m, 1H), 3.30-3.39 (m, 2H), 4.22-4.27(m, 1H), 6.96 (d, 1H, J=7.2 Hz), 7.27 (d, 1H, J=6.8 Hz), 7.68 (d, 1H,J=8.0 Hz), 8.41 (s, 1H), 8.65 (s, 1H) and 12.98 (brs, 1H); ¹³C NMR (100MHz, CDCl₃) δ 22.8, 24.0, 28.3, 32.9, 34.3, 35.0, 36.3, 42.9, 50.8,53.4, 53.7, 67.8, 123.9, 136.7, 139.9, 147.1, 148.3, 172.4 and 181.2;Mass spectrum (FAB+), m/z 346.2125 (M+H)⁺ (C₁₉H₂₈N₃O₃ requires m/z346.2131).

(S)-Methyl1-(6-(2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)piperidine-4-carboxylate(34)

To an ice-cool stirred solution of 7b (65 mg, 0.248 mmol) in dry DMF(0.7 mL) was added HBTU (141 mg, 0.372 mmol). After 10 min, a solutionof methyl piperidine-4-carboxylate (53 mg, 0.246 mmol) andN-methylmorpholine (82 μL, 0.744 mmol) in dry DMF (0.3 mL) was added at0° C. After 18 h at room temperature, the reaction mixture was dilutedwith water (20 mL), extracted with ethyl acetate (2×20 mL). The combinedorganic phase was washed with brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was further purifiedby flash chromatography on a silica gel column (15×2 cm) eluting with 5%methanol in CH₂Cl₂ afforded 34 as a pale yellow oil: yield 62 mg (65%);silica gel TLC R_(f) 0.35 (5% methanol in CH₂Cl₂); ¹H NMR (400 MHz,CDCl₃) δ 1.15-1.35 (m, 2H), 1.39-1.1.46 (m, 2H), 1.48-1.66 (m, 5H),1.81-1.91 (m, 4H), 2.05-2.20 (m, 3H), 2.25 (t, 2H, J=8.0 Hz), 2.41-2.54(m, 2H), 2.75 (t, 1H, J=12.0 Hz), 3.01-3.07 (m, 1H), 3.24 (s, 1H), 3.33(s, 1H), 3.67 (s, 3H), 3.75 (d, 1H, J=13.6 Hz), 4.37 (d, 1H, J=13.2 Hz),7.24 (s, 1H), 7.69 (s, 1H), 8.48 (s, 1H) and 8.53 (s, 1H); ¹³C NMR (100MHz, CDCl₃) δ 22.7, 25.3, 27.3, 28.0, 28.6, 33.3, 35.1, 40.9, 41.0,44.9, 51.9, 53.7, 54.3, 67.9, 123.8, 135.1, 148.6, 149.6, 171.4 and174.8; Mass spectrum (APCI+), m/z 388.2607 (M+H)⁺ (C₂₂H₃₄N₃O₃ requiresm/z 388.2600).

(S)-1-(6-(2-(Pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)piperidine-4-carboxylicacid (35)

A solution of 34 (41 mg, 0.106 mmol) in methanol (0.6 mL) was added aqNaOH (13 mg, 0.318 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, dissolved in 2 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×1 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded35 as a pale yellow oil: yield 28 mg (75%); silica gel TLC R_(f) 0.25(1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.17-1.26 (m, 2H),1.41-1.60 (m, 6H), 1.71-1.92 (m, 6H), 2.08-2.28 (m, 5H), 2.40-2.45 (m,2H), 2.74 (t, 2H), 3.02 (t, 2H), 3.30-3.35 (m, 2H), 3.70 (d, 1H), 4.36(d, 1H), 7.28 (s, 1H), 7.76 (d, 1H), 8.51 (d, 2H) and 10.12 (brs, 1H).¹³C NMR (100 MHz, CDCl₃) δ 22.4, 25.1, 27.1, 27.9, 28.2, 28.8, 33.1,34.5, 41.2, 45.1, 45.1, 53.3, 54.0, 67.7, 136.1, 147.6, 148.5 and 171.5;Mass spectrum (APCI+), m/z 374.2438 (M+H)⁺ (C₂₁H₃₂N₃O₃ requires m/z374.2444).

(S)-Methyl1-(6-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)pyrrolidine-3-carboxylate(36)

To an ice-cool stirred solution of 7b (50 mg, 0.191 mmol) in dry DMF(0.7 mL) was added HBTU (108 mg, 0.286 mmol). After 10 min, a solutionof (S)-methyl pyrrolidine-3-carboxylate (37 mg, 0.286 mmol) andN-methylmorpholine (63 μL, 0.573 mmol) in dry DMF (0.3 mL) was added at0° C. After 18 h at room temperature, the reaction mixture was dilutedwith water (20 mL), extracted with ethyl acetate (2×20 mL). The combinedorganic phase was washed with brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was further purifiedby flash chromatography on a silica gel column (15×1 cm) eluting with 5%methanol in CH₂Cl₂ afforded 36 as a pale yellow oil: yield 30 mg (42%);silica gel TLC R_(f) 0.35 (5% methanol in CH₂Cl₂); ¹H NMR (400 MHz,CDCl₃) δ 1.17-1.27 (m, 2H), 1.46-1.58 (m, 6H), 1.86-1.89 (m, 2H),2.00-2.25 (m, 8H), 2.38-2.50 (m, 1H), 2.51-2.54 (m, 1H), 2.97-3.00 (m,1H), 3.06-3.16 (m, 1H), 3.35-3.40 (m, 1H), 3.45-3.58 (m, 4H), 3.60-3.68(m, 4H (3+1)), 7.24 (d, 1H, J=6.0 Hz), 7.88 (s, 1H), 8.46 (d, 1H, J=4.8Hz) and 8.52 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.2, 24.3, 27.0, 27.7,29.1, 34.2, 41.6, 42.5, 45.0, 48.5, 52.3, 53.5, 53.9, 68.3, 123.8,135.7, 149.2, 149.8, 171.4 and 173.5; Mass spectrum (FAB+), m/z 374.2442(M+H)⁺ (C₂₁H₃₂N₃O₃ requires m/z 374.2444).

(S)-1-(6-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)pyrrolidine-3-carboxylicacid (37)

A solution of 36 (30 mg, 0.08 mmol) in methanol (0.6 mL) was added aqNaOH (9.7 mg, 0.24 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, dissolved in 2 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×1 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded37 as a pale yellow oil: yield 18 mg (62%); silica gel TLC R_(f) 0.25(1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.07-1.24 (m, 1H),1.28-1.91 (m, 7H), 1.92-2.40 (m, 8H), 2.93-3.11 (m, 1H), 3.31-3.48 (m,3H), 3.49-3.68 (m, 2H), 7.30 (dd, 1H, J=7.4, 8.2 Hz), 7.71 (d, 1H, J=7.8Hz), 8.45 (d, 1H, J=4.6 Hz) and 8.57 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ22.4, 24.9, 27.0, 27.8, 28.3, 33.8, 45.0, 46.0, 48.9, 49.5, 52.9, 53.1,67.8, 123.9, 136.6, 147.6, 148.8 and 171.7; Mass spectrum (APCI+), m/z360.2294 (M+H)⁺ (C₂₀H₃₀N₃O₃ requires m/z 360.2287).

(S)-Ethyl2-(4-(6-(2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)piperazin-1-yl)acetate(38)

To an ice-cool stirred solution of 7b (50 mg, 0.191 mmol) in dry DMF(0.7 mL) was added HBTU (108 mg, 0.286 mmol). After 10 min, a solutionof 19 (49 mg, 0.286 mmol) and N-methylmorpholine (63 μL, 0.573 mmol) indry DMF (0.3 mL) was added at 0° C. After 18 h at room temperature, thereaction mixture was diluted with water (20 mL), extracted with ethylacetate (2×20 mL). The combined organic phase was washed with brine,dried over anhydrous MgSO₄ and concentrated under diminished pressure.The residue was further purified by flash chromatography on a silica gelcolumn (15×2 cm) eluting with 10% methanol in CH₂Cl₂ afforded 38 as apale yellow oil: yield 42 mg, (53%); silica gel TLC R_(f) 0.17 (10%methanol in CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.16-1.40 (m, 5H),1.45-1.64 (m, 4H), 1.92 (brs, 2H), 2.06 (brs, 1H), 2.24-2.28 (m, 4H),2.39 (brs, 1H), 2.53-2.65 (m, 4H), 3.23 (s, 2H), 3.48 (m, 3H), 3.65 (t,2H, J=5.2 Hz), 4.19 (q, 2H, J=7.2, 14.4 Hz), 7.25-7.33 (dd, 1H, J=5.2,6.0 Hz), 7.76-7.97 (m, 1H), 8.52 (d, 1H, J=4.0 Hz) and 8.57 (d, 1H,J=1.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 14.3, 22.4, 25.0, 27.1, 33.0,41.4, 45.4, 52.6, 53.0, 53.6, 54.1, 59.2, 60.8, 123.7, 135.5, 149.8,170.0 and 171.4; Mass spectrum (APCI+), m/z 417.2868 (M+H)⁺ (C₂₃H₃₇N₄O₃requires m/z 417.2866).

(S)-2-(4-(6-(2-(Pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)piperazin-1-yl)aceticacid (39)

A solution of 38 (40 mg, 0.10 mmol) in methanol (0.6 mL) was added aqNaOH (12 mg, 0.29 mmol) with stirring at room temperature. After 18 h,the reaction mixture was evaporated, dissolved in 1 mL of acetone andthe pH was adjusted to 7 by using acetic acid. Acetone was evaporatedand the crude product was directly purified by flash chromatography on asilica gel column (10×1 cm), eluting with 1:1.5 methanol-CH₂Cl₂ afforded39 as a pale yellow oil: yield 19 mg (49%); silica gel TLC R_(f) 0.2(1:1.5 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.16-1.36 (m, 2H),1.39-1.54 (m, 4H), 1.63-1.71 (m, 1H), 1.79-1.87 (m, 1H), 1.90-1.96 (m,1H), 2.04-2.11 (m, 1H), 2.13-2.26 (m, 4H), 2.41-2.48 (m, 5H), 3.00 (brs,2H), 3.27 (t, 1H, J=8.4 Hz), 3.33 (t, 1H, J=7.6 Hz), 3.47 (brs, 2H),3.61 (brs, 2H), 7.23-7.26 (dd, 1H, J=4.8, 7.2 Hz), 7.69 (d, 1H, J=8.0Hz), 8.46 (d, 1H, J=3.6 Hz) and 8.54 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ22.6, 25.1, 27.2, 28.5, 32.0, 35.0, 40.8, 44.8, 53.0, 53.5, 54.2, 67.7,123.6, 135.3, 139.6, 148.2, 149.2 and 171.4; Mass spectrum (APCI+), m/z389.2563 (M+H)⁺ (C₂₁H₃₃N₄O₃ requires m/z 389.2553).

(S)-Allyl1-(6-(2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)-1H-pyrrole-3-carboxylate(40)

To a stirred solution of (S)-6-(2-(pyridin-3-yl)pyrrolidin-1-yl)hexanoicacid (7b, 72 mg, 0.275 mmol) and a drop of DMF in dry CH₂Cl₂ (2 mL) wasadded oxalyl chloride (47 μL, 0.550 mmol). After 2 h at roomtemperature, reaction mixture was concentrated under reduced pressureyielded the acid chloride. This acid chloride was dissolved in DCM (2mL) and added drop wise to a stirred solution containing allyl1H-pyrrole-3-carboxylate (21, 46 mg, 0.30 mmol), DMAP (19 mg, 0.152mmol) and diisopropylethylamine (159 μL, 0.913 mmol) in dry CH₂Cl₂ (5mL). The reaction mixture was stirred for 18 h at room temperature. Thereaction mixture was diluted with CH₂Cl₂ (20 mL), washed with aq satdNaHCO₃ (10 mL), water, brine, dried over anhydrous MgSO₄ andconcentrated under diminished pressure. The residue was purified by aflash chromatography on a silica gel column (20×2 cm) eluting with 1:19methanol-CH₂Cl₂ afforded 40 as a pale yellow oil: yield 54 mg (50%);silica gel TLC R_(f) 0.31 (1:19 methanol-CH₂Cl₂); ¹H NMR (400 MHz,CDCl₃) δ 1.27-1.50 (m, 4H), 1.62-1.74 (m, 3H), 1.80-1.87 (m, 1H),1.89-1.97 (m, 1H), 2.06-2.25 (m, 3H), 2.44-2.51 (m, 1H), 2.79 (t, 2H,J=7.6 Hz), 3.25 (t, 1H, J=8.0 Hz), 3.33 (t, 1H, J=6.4 Hz), 4.75 (t, 2H,J=6.0 Hz), 5.27 (d, 1H, J=12.0 Hz), 5.37 (d, 1H, J=17.2 Hz), 5.96-6.05(m, 1H), 6.68 (s, 1H), 7.23-7.29 (m, 2H), 7.69 (d, 1H, J=7.6 Hz), 7.88(s, 1H), 8.49 (s, 1H) and 8.55 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.7,24.2, 26.8, 28.5, 34.4, 35.2, 53.7, 54.1, 65.0, 67.7, 112.9, 118.2,119.7, 120.2, 123.7, 132.4, 134.9, 148.5, 149.5, 163.6 and 170.4; Massspectrum (APCI+), m/z 396.2280 (M+H)⁺ (C₂₃H₃₀N₃O₃ requires m/z396.2287).

(S)-1-(6-(2-(Pyridin-3-yl)pyrrolidin-1-yl)hexanoyl)-1H-pyrrole-3-carboxylicacid (41)

To a stirred solution of 40 (52 mg, 0.132 mmol) in dry CH₂Cl₂ (5 mL) wasadded phenyl silane (243 μL, 1.97 mmol) and Pd(PPh₃)₄ (15 mg, 0.013mmol) at room temperature. After 1 h, the reaction mixture wasconcentrated under diminished pressure and the residue was directlypurified by flash chromatography on a silica gel column (15×1 cm)eluting 1:1.5 methanol-CH₂Cl₂ afforded 41 as a pale yellow oil: yield 31mg (66%); silica gel TLC R_(f) 0.28 (1:1.5 methanol-CH₂Cl₂); ¹H NMR (400MHz, CDCl₃) δ 1.25-1.45 (m, 4H), 1.51-1.65 (m, 2H), 1.66-1.77 (m, 1H),1.78-1.88 (m, 1H), 1.89-2.03 (m, 1H), 2.05-2.24 (m, 2H), 2.26-2.34 (m,1H), 2.37-2.51 (m, 1H), 2.58-2.80 (m, 2H), 3.25-3.43 (m, 2H), 6.59 (s,1H), 7.18 (s, 1H), 7.29 (s, 1H), 7.78 (s, 1H), 7.79 (s, 1H), 8.51 (s,1H), 8.61 (s, 1H) and 11.89 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.6,24.0, 26.6, 27.9, 34.1, 34.7, 53.4, 53.9, 67.8, 113.3, 119.1, 123.5,123.8, 135.9, 139.1, 147.9, 148.8 and 170.5; Mass spectrum (APCI+), m/z356.1967 (M+H)⁺ (C₂₀H₂₆N₃O₃ requires m/z 356.1974).

tert-Butyl 4-((4-hydroxybutyl)carbamoyl)piperidine-1-carboxylate (43)

To an ice-cool stirred solution of 42 (650 mg, 2.84 mmol) in dry THF (15mL) was added trimethylamine (1.18 mL, 8.49 mmol) andN,N′-disuccinimidyl carbonate (1.14 g, 4.25 mmol). The reaction mixturewas stirred at room temperature for 3 h and filtered. To the filtratewas added 4-aminobutan-1-ol (378 mg, 4.25 mmol) and stirred for 3 h. Thereaction mixture was diluted with CH₂Cl₂ (100 mL), washed with water,brine, dried over MgSO₄ and concentrated under diminished pressure. Theresidue was purified by chromatography on a silica gel column (20×4 cm)eluting with 1:19 methanol-CH₂Cl₂ afforded 43 as a colorless oil: yield690 mg (81%); silica gel TLC R_(f) 0.27 (1:19 methanol-CH₂Cl₂); ¹H NMR(400 MHz, CDCl₃) δ 1.45 (s, 9H), 1.54-1.76 (m, 6H), 1.77-1.81 (m, 1H),2.19-2.27 (m, 1H), 2.73 (t, 2H, J=11.6 Hz), 2.90 (brs, 1H), 3.28 (q, 2H,J=6.4, 12.4 Hz), 3.66 (t, 2H, J=6.0 Hz), 4.12 (d, 2H, J=9.2 Hz) and 6.25(t, 1H, J=5.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 26.3, 28.4, 28.7, 29.7,39.2, 43.3, 62.1, 79.7, 154.7 and 174.7; Mass spectrum (FAB+), m/z301.2134 (M+H)⁺ (C₁₅H₂₉N₂O₄ requires m/z 301.2127).

tert-Butyl 4-((5-hydroxypentyl)carbamoyl)piperidine-1-carboxylate (44)

To an ice-cool stirred solution of 42 (580 mg, 2.53 mmol) in dry THF (15mL) was added trimethylamine (1.05 mL, 7.59 mmol) andN,N′-disuccinimidyl carbonate (971 mg, 3.79 mmol). The reaction mixturewas stirred at room temperature for 3 hours and filtered. To thefiltrate was added 5-aminopentan-1-ol (390 mg, 3.79 mmol) and stirredfor 3 h. The reaction mixture was diluted with CH₂Cl₂ (100 mL), washedwith water, brine, dried over MgSO₄ and concentrated under diminishedpressure. The crude product was purified by chromatography on silica gelcolumn (20×4 cm) by eluting with DCM/methanol (19:1) to give 44 as acolorless oil: yield 384 mg (80%); silica gel TLC R_(f) 0.27 (5% MeOH inCH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 1.34-1.42 (m, 2H), 1.45 (s, 9H),1.48-1.66 (m, 6H), 1.77 (d, 2H, J=11.2 Hz), 2.25-232 (m, 1H), 2.71-2.75(m, 3H), 3.22 (q, 2H, J=6.8, 13.2 Hz), 3.60 (t, 2H, J=6.4 Hz), 4.11 (d,2H, J=12.0 Hz) and 6.55 (t, 1H, J=5.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ23.0, 25.4, 28.4, 28.6, 29.1, 32.0, 39.3, 43.1, 62.2, 79.8, 154.8 and175.1.

tert-Butyl4-((4-((methylsulfonyl)oxy)butyl)carbamoyl)piperidine-1-carboxylate (45)

To an ice-cool stirred solution of 43 (400 mg, 1.33 mmol) in dry CH₂Cl₂(5 mL) was added triethylamine (0.56 mL, 3.99 mmol) and methanesulfonylchloride (0.16 mL, 2.00 mmol). After 2 hours at room temperature,reaction mixture was diluted with CH₂Cl₂ (40 mL), washed with satd aqNaHCO₃, water, brine, dried over anhydrous MgSO₄ and concentrated underdiminished pressure. The residue was purified by chromatography on asilica gel column (15×2 cm) eluting with 4:1 ethyl acetate-hexanesafforded 45 as a colorless oil: yield 430 mg (85%); silica gel TLC R_(f)0.56 (19:1 dichloromethane-methanol); ¹H NMR (400 MHz, CDCl₃) δ 1.45 (s,9H), 1.60-1.67 (m, 4H), 1.74-1.80 (m, 4H), 2.22-2.28 (m, 1H), 2.73 (brs,2H), 3.03 (s, 3H), 3.28 (q, 2H, J=6.8, 12.8 Hz), 4.13 (brs, 2H), 4.25(t, 2H, J=6.4 Hz) and 6.18 (t, 1H, J=5.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ25.7, 26.5, 28.4, 28.6, 37.3, 38.5, 43.2, 69.8, 79.6, 154.7 and 174.7;Mass spectrum (APCI+), m/z 379.1910 (M+H)⁺ (C₁₆H₃₁N₂O₆S requires m/z379.1903).

tert-Butyl4-((5-((methylsulfonyl)oxy)pentyl)carbamoyl)piperidine-1-carboxylate(46)

To a solution of compound 44 (380 mg, 1.21 mmol) in dichloromethane (5mL) was added triethylamine (422 μL, 3.02 mmol) and methanesulfonylchloride (122 μL, 1.57 mmol) at 0° C. The reaction mixture was warmed toroom temperature, stirred for 2 hours, and extracted withdichloromethane and water. The organic layer was washed with sat. NaHCO₃solution, brine, dried over anhydrous magnesium sulfate andconcentrated. The crude was purified through chromatography with silicagel eluting with 1:4 hexanes-ethyl acetate to give compound 46 as a paleyellow oil; yield 426 mg (90%); silica gel TLC R_(f) 0.56 (19:1dichloromethane-methanol); ¹H NMR (400 MHz, CDCl₃) δ 1.42-1.48 (m, 9+2H, 1.52-1.58 (m, 2H), 1.60-1.67 (m, 2H), 1.75-1.81 (m, 4H), 2.22-2.28(m, 1H), 2.72 (d, 2H, J=10.4 Hz), 3.02 (s, 3H), 3.24 (q, 2H, J=6.8, 12.8Hz), 4.12 (d, 2H, J=10.0 Hz), 4.23 (t, 2H, J=6.0 Hz) and 6.15 (t, 1H,J=6.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 22.7, 28.4, 28.6, 28.7, 28.9,37.3, 38.9, 43.1, 70.0, 79.5, 154.7 and 174.6.

tert-Butyl 4-((4-iodobutyl)carbamoyl)piperidine-1-carboxylate (47)

A mixture of 45 (430 mg, 1.14 mmol) and sodium iodide (750 mg, 5.68mmol) in acetonitrile (2 mL) was vigorously stirred at room temperature.After 18 hours, the mixture was evaporated and the residue waspartitioned between ethyl acetate and water. The organic phase waswashed with aq Na₂S₂O₃ solution, water, brine, dried over MgSO₄ andconcentrated under diminished pressure. The residue was purified bychromatography on a silica gel column (15×2 cm) eluting with 1:1 ethylacetate-hexanes afforded 47 as a pale yellow oil (290 mg, 62% yield);silica gel TLC R_(f) 0.66 (4:1 ethyl acetate-hexanes); ¹HNMR (400 MHz,CDCl₃) δ 1.45 (s, 9H), 1.58-1.67 (m, 4H), 1.77-1.86 (m, 4H), 2.20-2.27(m, 1H), 3.20 (t, 2H, J=6.8 Hz), 3.25-3.30 (q, 2H, J=6.8, 13.2 Hz), 4.13(d, 2H, J=6.4 Hz) and 5.94 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 6.2,28.4, 28.7, 30.5, 30.6, 38.2, 43.3, 79.6, 154.6 and 174.5; Mass spectrum(APCI+), m/z 411.1148 (M+H)⁺ (C₁₅H₂₈N₂O₃I requires m/z 411.1145).

tert-Butyl 4-((5-iodopentyl)carbamoyl)piperidine-1-carboxylate (48)

A mixture of 46 (420 mg, 1.07 mmol) and sodium iodide (802 mg, 5.35mmol) in acetonitrile (6 mL) was vigorously stirred at room temperature.After 18 hours, the mixture was evaporated and the residue waspartitioned between ethyl acetate and water. The EtOAc layer was washedwith Na₂S₂O₃ solution, water, brine, dried over MgSO₄, and evaporated.The residue was purified by chromatography on silica gel eluting with3:2 hexanes-ethyl acetate to give 48 as a pale yellow oil: yield 293 mg(65%); silica gel TLC R_(f) 0.66 (1:4 hexanes-ethyl acetate); ¹H NMR(400 MHz, CDCl₃) δ 1.38-1.45 (m, 9+2 H, 1.49-1.55 (m, 2H), 1.61-1.69 (m,2H), 1.77-1.86 (m, 4H), 2.24-2.28 (m, 1H), 2.26 (d, 2H), 3.19 (t, 2H,J=7.2 Hz), 3.24 (q, 2H, J=6.8, 12.8 Hz), 4.12 (d, 2H, J=8.0 Hz) and 6.16(t, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 6.9, 27.6, 28.4, 28.5, 28.6, 32.8,39.0, 43.2, 79.6, 154.6 and 174.5.

(S)-tert-Butyl4-((4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butyl)carbamoyl)piperidine-1-carboxylate(49)

A solution of 47 (198 mg, 0.48 mmol) in acetonitrile (0.3 mL) was addedto a stirred solution of 5 (65 mg, 0.44 mmol) and diisopropylethylamine(153 μL, 0.88 mmol) in acetonitrile (0.8 mL) at room temperature. After18 hours, reaction mixture was concentrated under diminished pressure,residue was directly purified by flash chromatography on a silica gelcolumn (15×2 cm) eluting with 10% methanol in CH₂Cl₂ afforded 49 as apale yellow oil: yield 95 mg (51%); silica gel TLC R_(f) 0.16 (9:1dichloromethane-methanol); ¹H NMR (400 MHz, CDCl₃) δ 1.38-1.65 (m,9H+6H), 1.75-1.78 (m, 3H), 1.87-1.91 (m, 1H), 1.93-2.01 (m, 1H),2.15-2.33 (m, 4H), 2.50-2.53 (m, 1H), 2.73 (t, 2H, J=12.0 Hz), 3.13-3.17(m, 2H), 3.42 (brs, 2H), 4.12 (brs 2H), 5.76 (s, 1H), 7.26 (d, 1H, J=6.0Hz), 7.79 (s, 1H), 8.51 (d, 1H, J=3.6 Hz) and 8.56 (d, 1H, J=1.6 Hz);¹³C NMR (100 MHz, CDCl₃) δ 22.5, 25.6, 27.4, 28.4, 28.7, 28.7, 34.7,39.0, 43.4, 53.5, 53.84, 79.6, 123.6, 135.3, 148.9, 149.6, 154.7 and174.4; Mass spectrum (APCI+), m/z 431.3024 (M+H)⁺ (C₂₄H₃₉N₄O₃ requiresm/z 431.3022).

(S)-tert-Butyl4-((5-(2-(pyridin-3-yl)pyrrolidin-1-yl)pentyl)carbamoyl)piperidine-1-carboxylate(50)

A solution of 48 (241 mg, 0.567 mmol) in acetonitrile (0.5 mL) was addedto a mixture of 5 (70 mg, 0.472 mmol) and diisopropylethylamine (206 μL,1.18 mmol) in acetonitrile (1 mL) with stirring at room temperature.After 18 hours, the mixture was evaporated and the residue was purifiedby chromatography on silica gel eluting with 9:1dichloromethane-methanol to give product 50 as a pale yellow oil: yield102 mg (49%); silica gel TLC R_(f) 0.16 (9:1 dichloromethane-methanol);¹H NMR (500 MHz, CDCl₃) δ 1.15-1.23 (m, 1H), 1.24-1.35 (m, 1H),1.39-1.49 (m, 13H (9+4)), 1.77 (t, 3H, J=9.6 Hz), 1.84-1.96 (m, 1H),1.97-2.08 (m, 1H), 2.19-2.34 (m, 3H), 2.36-2.46 (m, 1H), 2.48-2.58 (m,1H), 2.65-2.81 (m, 2H), 3.18 (d, 2H, J=5.8 Hz), 3.24 (d, 1H, J=7.3 Hz),3.45-3.61 (m, 3H), 3.79-3.83 (m, 1H), 4.11 (brs, 2H), 6.19 (s, 1H), 7.29(s, 1H), 7.83 (s, 1H), 8.50 (s, 1H), 8.59 (s, 1H); ¹³C NMR (125 MHz,CDCl₃) δ 12.4, 22.5, 24.5, 27.7, 28.4, 28.6, 29.2, 34.6, 39.1, 43.1,53.5, 54.8, 68.0, 79.4, 123.7, 135.4, 148.8, 149.6, 154.6 and 174.5.

(S)-Ethyl2-(4-((4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butyl)carbamoyl)piperidin-1-yl)acetate(51)

To an ice-cooled stirred solution of 49 (95 mg, 0.22 mmol) in dry CH₂Cl₂(2 mL) was added trifluoroacetic acid (0.25 mL, 3.31 mmol). After 18hours at room temperature, reaction mixture was co-evaporated with 1 mLof toluene (3 times) yielded(S)—N-(4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butyl)piperidine-4-carboxamide,which was directly used for the next reaction without any furtherpurification. A solution of ethyl iodoacetate (71 mg, 0.33 mmol) inacetonitrile (0.2 mL) was added to a stirred solution of(S)—N-(4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butyl)piperidine-4-carboxamidein acetonitrile (2 mL) and DIPEA (157 μL, 0.90 mmol) at roomtemperature. After 18 h, the reaction mixture was evaporated underdiminished pressure. The residue was purified by flash chromatography ona silica gel column (15×1 cm) eluting with 1:9 methanol-CH₂Cl₂ afforded51 as a pale yellow oil: yield 21 mg (28%); silica gel TLC R_(f) 0.12(9:1 dichloromethane-methanol); ¹H NMR (400 MHz, CDCl₃) δ 1.27 (t, 3H,J=7.2 Hz), 1.36-1.51 (m, 4H), 1.70-1.88 (m, 6H), 1.93-1.99 (m, 1H),2.02-2.09 (m, 1H), 2.13-2.26 (m, 5H), 2.43-2.48 (m, 1H), 2.97 (d, 2H,J=11.2 Hz), 3.13-3.19 (q, 2H, J=7.2, 14.4 Hz), 3.27-3.37 (m, 2H),4.15-4.21 (q, 2H, J=7.6, 14.4 Hz), 5.66 (s, 1H), 7.25 (d, 1H, J=7.2 Hz),7.72 (d, 1H, J=6.8 Hz), 8.50 (s, 1H) and 8.55 (s, 1H); ¹³C NMR (100 MHz,CDCl₃) δ 14.2, 22.6, 25.9, 27.5, 28.9, 35.0, 39.1, 42.9, 52.8, 53.5,53.9, 59.7, 60.5, 67.9, 123.7, 135.0, 148.6, 149.5, 170.4 and 174.7;Mass spectrum (FAB+), m/z 417.2880 (M+H)⁺ (C₂₃H₃₇N₄O₃ requires m/z417.2866).

(S)-Ethyl2-(4-((5-(2-(pyridin-3-yl)pyrrolidin-1-yl)pentyl)carbamoyl)piperidin-1-yl)acetate(52)

To a stirred solution of 50 (100 mg, 0.225 mmol) in dry DCM was addedTFA (258 μL, 3.37 mmol). After 3 h the reaction mixture wasco-evaporated with toluene (3 times) to give boc deprotected compound,which was directly used for the next reaction. A solution of ethyliodoacetate (128 mg, 0.331 mmol) in acetonitrile (0.2 mL) was added to astirred solution of boc deprotected compound and DIPEA (157 μL, 0.90mmol) in acetonitrile (2 mL). After 18 hours, the reaction mixture wasevaporated under diminished pressure. The residue was directly purifiedby chromatography on silica gel column (15×1 cm) eluting with 9:1dichloromethane-methanol to give compound 52 as a pale yellow oil: yield36 mg (37%); silica gel TLC R_(f) 0.12 (9:1 dichloromethane-methanol);¹H NMR (500 MHz, CDCl₃) δ 1.11-1.25 (m, 6H), 1.29-1.35 (m, 2H), 1.41(brs, 2H), 1.69-1.83 (m, 5H), 1.99-2.04 (m, 2H), 2.16-2.19 (m, 3H),2.27-2.29 (m, 1H), 2.46 (brs, 1H), 2.90 (d, 2H, J=11.2 Hz), 3.09-3.12(m, 2H), 3.14 (s, 2H), 3.37 (brs, 2H), 4.08-4.13 (m, 3H), 5.59 (s, 1H),7.23 (s, 1H), 7.77 (s, 1H), 8.45 (s, 1H) and 8.49 (s, 1H); ¹³C NMR (125MHz, CDCl₃) δ 13.2, 21.4, 27.8, 28.2, 28.7, 38.1, 41.8, 51.8, 52.5,52.8, 58.6, 59.5, 122.7, 134.3, 148.7, 169.4 and 173.7;

(S)-2-(4-((4-(2-(Pyridin-3-yl)pyrrolidin-1-yl)butyl)carbamoyl)piperidin-1-yl)aceticacid (53)

A solution of 51 (20 mg, 0.048 mmol) in methanol (0.5 mL) was added aqNaOH (6 mg, 0.144 mmol) with stirring at room temperature. After 18hours, the reaction mixture was evaporated, dissolved in 1 mL of acetoneand the pH was adjusted to 7 by using acetic acid. Acetone wasevaporated and the crude product was directly purified by flashchromatography on a silica gel column (10×1 cm), eluting with 1:1methanol-CH₂Cl₂ afforded 53 as a pale yellow oil: yield 13 mg (66%);silica gel TLC R_(f) 0.12 (1:1 methanol-CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃)δ 1.20-1.51 (m, 4H), 1.52-1.61 (m, 1H), 1.62-2.00 (m, 7H), 2.12-2.36 (m,6H), 3.03-3.23 (m, 8H), 7.18 (s, 1H), 7.47 (brs, 1H), 7.61 (d, 1H, J=7.2Hz), 8.38 (d, 1H, J=2.0 Hz) and 8.46 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ22.8, 26.4, 27.1, 27.6, 35.3, 39.4, 52.7, 53.6, 54.1, 67.7, 123.7,135.2, 139.9, 148.4, 149.3 and 174.6; Mass spectrum (FAB+), m/z 389.2552(M+H)⁺ (C₂₁H₃₃N₄O₃ requires m/z 389.2553).

(S)-2-(4-((5-(2-(Pyridin-3-yl)pyrrolidin-1-yl)pentyl)carbamoyl)piperidin-1-yl)aceticacid (54)

A solution of 52 (34 mg, 0.079 mmol) in methanol (1 mL) was added aqNaOH (9.5 mg, 0.237 mmol) with stirring at room temperature. After 18hours, the reaction mixture was evaporated, dissolved in acetone (3 mL)and the pH was adjusted to 7 by using acetic acid. Acetone wasevaporated and the crude product was purified by chromatography onsilica gel column (10×1 cm), eluting with DCM/methanol (1:1) to give 54as a pale yellow oil: yield 14 mg (44%); silica gel TLC R_(f) 0.12 (1:1methanol-dichloromethane); ¹H NMR (400 MHz, CDCl₃) δ 1.09-1.26 (m, 2H),1.32-1.35 (m, 4H), 1.55-1.62 (m, 1H), 1.74-1.79 (m, 1H), 1.80-1.88 (m,1H), 1.90-1.99 (m, 3H), 2.05-2.16 (m, 4H), 2.32-2.39 (m, 2H), 2.83 (brs,2H), 3.07 (brs, 2H), 3.16-3.26 (m, 2H), 3.43 (brs, 2H), 3.50 (brs, 2H),7.17 (dd, 1H, J=4.4, 7.6 Hz), 7.31 (brs, 1H), 7.61 (d, 1H, J=8.0 Hz),8.39 (s, 1H) and 8.47 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.8, 24.8,26.2, 28.5, 29.4, 35.3, 39.6, 52.7, 53.6, 54.3, 59.6, 67.7, 123.7,135.3, 139.9, 148.4, 149.4, 168.6 and 173.7.

We claim:
 1. A method for obtaining a nicotine hapten capable ofeliciting an immune response specific to nicotine, the method comprising(a) providing three-dimensional structural information of an immunogeniccarrier; (b) selecting functional groups or small molecule fragmentspredicted to bind to free nicotine at an immunogenic carrier bindingsite, wherein a functional group or small molecule fragment is selectedif indicated to exhibit higher binding energy for free nicotine than forthe immunogenic carrier; and (c) linking the selected functional groupor small molecule fragment in a single compound, wherein the compound isa nicotine hapten that, when conjugated to the immunogenic carrier,elicits production of anti-nicotine antibodies having an affinity indexgreater than 0.02 and less than or equal to
 1. 2. The method of claim 1,wherein selecting comprises using a computer having a non-transitorycomputer-readable storage medium containing programming to perform afitting operation and to determine one or more binding energy parametersbetween nicotine and a binding site of the immunogenic carrier.
 3. Themethod of claim 2, further comprising analyzing results of the fittingoperation to characterize the association between nicotine and thebinding pocket.
 4. The method of claim 1, further comprising: (d)synthesizing or obtaining the compound; and (e) evaluating the compoundfor its ability to compete with free nicotine for binding.
 5. The methodof claim 4, wherein evaluating comprises performing an indirectcompetitive enzyme-linked immunosorbent assay (ELISA).
 6. The method ofclaim 1, wherein the hapten comprises nicotine or a nicotine derivative.7. The method of claim 1, wherein the immunogenic carrier is astreptavidin.